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The folding pathway of the genomic hepatitis delta virus ribozyme is dominated by slow folding of the pseudoknots
Abstract
Hepatitis delta virus (HDV) replicates by a double rolling-circle mechanism that requires self-cleavage by closely related genomic and antigenomic versions of a ribozyme. We have previously shown that the uncleaved genomic ribozyme is subject to a variety of alternative (Alt) pairings. Sequence upstream of the ribozyme can regulate self-cleavage activity by formation of an Alt 1 ribozyme-containing structure that severely inhibits self-cleavage, or a P(-1) self-structure that permits rapid self-cleavage. Here, we test three other alternative pairings: Alt P1, Alt 2, and Alt 3. Alt P1 and Alt 3 contain primarily ribozyme-ribozyme interactions, while Alt 2 involves ribozyme-flanking sequence interaction. A number of single and double mutant ribozymes were prepared to increase or decrease the stability of the alternative pairings, and rates of self-cleavage were determined. Results of these experiments were consistent with the existence of the proposed alternative pairings and their ability to inhibit the overall rate of native ribozyme folding. Local misfolds are treated as internal equilibrium constants in a binding polynomial that modulates the intrinsic rate of cleavage. This model of equilibrium effects of misfolds should be general and apply to other ribozymes. All of the alternative pairings sequester a pseudoknot-forming strand. Folding of ribozymes containing Alt P1 and Alt 2 was accelerated by urea as long as the native ribozyme fold was sufficiently stable, while folding of mutants in which both of these alternative pairings had been removed were not stimulated by urea. This behavior suggests that the pseudoknots form by capture of an unfolded or appropriately rearranged alternative pairing by its complementary native strand. Fast-folding mutants were prepared by either weakening alternative pairings or by strengthening native pairings. A kinetic model was developed that accommodates these features and explains the position of the rate-limiting step for the G11C mutant. Implications of these results for structural and dynamic studies of the uncleaved HDV ribozyme are discussed.
... As self-cleaving catalytic RNA, the nascent HDV sequence undergoes self-cleavage during its rolling-circle replication process by forming a catalytic fold. [59][60][61] This native fold (state N in Fig. 2), which directly controls the self-cleaving activities, has a complex doublepseudoknot topology with several helices. Early experiment suggested that the self-cleavage of HDV in vitro is bi-phasic: about 30% RNAs fold into the native structure N in around 15 s and the rest slowly cleavages in the next 30 minute (min). ...
... Early experiment suggested that the self-cleavage of HDV in vitro is bi-phasic: about 30% RNAs fold into the native structure N in around 15 s and the rest slowly cleavages in the next 30 minute (min). [60] Refolding behaviors of the wildtype ribozyme show two distinguish stages as well, and these special features are further studied by recursive searching the states with high net flux-in (out) to identify the detailed folding pathway. [46] The results (see Fig. 2(a)) suggest that, the slow cleavages result from that part of the ribozymes trapped in the non-native state I1. ...
... Thus, even the rates from I1 to 863 and 863 to 639 are around 10 1 s −1 and 10 3 s −1 , the overall slow folding pathway is still limited by the transition from state I1 to 863. The non-phasic feature observed in mutated HDV folding experiments, [60] is because the mutation breaks GC pair and destabilizes I1, thereby decreasing the population flowing through I1. ...
RNAs carry out diverse biological functions, partly because different conformations of the same RNA sequence can play different roles in cellular activities. To fully understand the biological functions of RNAs requires a conceptual framework to investigate the folding kinetics of RNA molecules, instead of native structures alone. Over the past several decades, many experimental and theoretical methods have been developed to address RNA folding. The helix-based RNA folding theory is the one which uses helices as building blocks, to calculate folding kinetics of secondary structures with pseudoknots of long RNA in two different folding scenarios. Here, we will briefly review the helix-based RNA folding theory and its application in exploring regulation mechanisms of several riboswitches and self-cleavage activities of the hepatitis delta virus (HDV) ribozyme.
... The folding pathways of large functional RNAs have proven to be quite complex with intermediates that can be trapped for minutes to hours (Banerjee & Turner, 1995;Chadalavada et al., 2002;Zarrinkar et al., 1996). For example, 90% of the Tetrahymena ribozyme is found in a misfolded state that transitions to the native state with hour timescale kinetics (Banerjee & Turner, 1995), and the HDV ribozyme folds through numerous intermediates, some long-lived (Chadalavada et al., 2002). ...
... The folding pathways of large functional RNAs have proven to be quite complex with intermediates that can be trapped for minutes to hours (Banerjee & Turner, 1995;Chadalavada et al., 2002;Zarrinkar et al., 1996). For example, 90% of the Tetrahymena ribozyme is found in a misfolded state that transitions to the native state with hour timescale kinetics (Banerjee & Turner, 1995), and the HDV ribozyme folds through numerous intermediates, some long-lived (Chadalavada et al., 2002). Long-lived misfolded intermediates are often very similar in structure to the native RNA and typically arise from a secondary structure mispairing or an incorrect three-dimensional topology (Mitchell et al., 2013;Treiber et al., 1998;Wan et al., 2010). ...
... In the absence of crowding, physiological concentrations of Mg 2+ are not high enough to fold functional RNAs in a twostate manner. This is apparent from the observation of long-lived intermediates and slow folding under these conditions (Banerjee & Turner, 1995;Chadalavada et al., 2002;Mitchell et al., 2013). However, in the presence of biological crowding conditions and physiological Mg 2+ , functional RNAs tend to fold in a cooperative manner into compact structures (Desai et al., 2014;Dupuis et al., 2014;Strulson et al., 2014;Tyrrell et al., 2015), and ribozymes and riboswitches tend to have higher rates of cleavage and higher ligand binding affinity (Paudel & Rueda, 2014). ...
Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions
in vivo
. Within the last few years, methods have been developed to probe RNA structures
in vivo
and genome-wide. These studies reveal that RNA often adopts very different structures
in vivo
and
in vitro
, and provide profound insights into RNA biology. Nonetheless, both
in vitro
and
in vivo
approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies
in vitro
often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding
in vivo
. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such
in vivo-like
conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding
in vitro
and
in vivo
and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.
... 13 As a follow-up study, an equilibrium model is proposed comprising two intermediate and the native fold, which is confirmed by mutagenesis and kinetic characterization. 84 In this model, the 5 0 portion of P2 can base-pair with either the native 3 0 portion or nonnative ribozyme sequence made of nucleotides from P1, P3 and single-stranded regions. 84 Given that P2 has a driving role in the correct folding of the HDV ribozyme, the direction of the shifting of this equilibrium may explain the resultant ribozyme activity. ...
... 84 In this model, the 5 0 portion of P2 can base-pair with either the native 3 0 portion or nonnative ribozyme sequence made of nucleotides from P1, P3 and single-stranded regions. 84 Given that P2 has a driving role in the correct folding of the HDV ribozyme, the direction of the shifting of this equilibrium may explain the resultant ribozyme activity. 84 ...
... 84 Given that P2 has a driving role in the correct folding of the HDV ribozyme, the direction of the shifting of this equilibrium may explain the resultant ribozyme activity. 84 ...
Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All transient and dominant structures are mapped to our new alignments introduced here.
... The G d U wobble pair at bp 1 is in red (throughout) and boxed, while C75 is blue (throughout) and is boxed. The G11C mutation that promotes fast, single-exponential kinetics is shown (Chadalavada et al. 2002). (B) Crystal structure of the self-cleaved form of the ribozyme. ...
... In an effort to clarify what roles, if any, the GdU wobble pair plays in the HDV ribozyme reaction, constructs with the purine-pyrimidine combinations G d U, A-U, G-C, and A + d C at bp 1 were studied, and rate-pH and rate-Mg 2+ profiles were determined. In order to minimize the impact of alternative folding on activity and therefore study effects on chemistry, we used RNA sequences based on the previously characterized G11C fast-folding ribozymes (Chadalavada et al. , 2002. Ultimately, our data suggest that, in contrast to previous results, bp 1 does not need to be a wobble pair and that it provides a structural role in catalysis. ...
... The iterated loop matching (ILM) algorithm was used, as it is much less timeconsuming than other programs such as PKNOTS but has similar accuracy (Ruan et al. 2004a,b). All predictions were carried out in the background of a G11C mutation that, in combination with an antisense oligonucleotide, facilitates fast-folding of the À30/99 ribozyme (see next paragraph and Materials and Methods) (Chadalavada et al. , 2002. ...
The hepatitis delta virus (HDV) ribozyme occurs in the genomic and antigenomic strands of the HDV RNA and within mammalian transcriptomes. Previous kinetic studies suggested that a wobble pair (G*U or A(+)*C) is preferred at the cleavage site; however, the reasons for this are unclear. We conducted sequence comparisons, which indicated that while G*U is the most prevalent combination at the cleavage site, G-C occurs to a significant extent in genomic HDV isolates, and G*U, G-C, and A-U pairs are present in mammalian ribozymes. We analyzed the folding of genomic HDV ribozymes by free energy minimization and found that variants with purine-pyrimidine combinations at the cleavage site are predicted to form native structures while pyrimidine-purine combinations misfold, consistent with earlier kinetic data and sequence comparisons. To test whether the cleavage site base pair contributes to catalysis, we characterized the pH and Mg(2+)-dependence of reaction kinetics of fast-folding genomic HDV ribozymes with cleavage site base pair purine-pyrimidine combinations: G*U, A-U, G-C, and A(+)*C. Rates for these native-folding ribozymes displayed highly similar pH and Mg(2+) concentration dependencies, with the exception of the A(+)*C ribozyme, which deviated at high pH. None of the four ribozymes underwent miscleavage. These observations support the A(+)*C ribozyme as being more active with a wobble pair at the cleavage site than with no base pair at all. Overall, the data support a model in which the cleavage site base pair provides a structural role in catalysis and does not need to be a wobble pair.
... In vivo, during the transcription elongation, because the upstream has already been folded, this will further influence the folding pathways of the downstream section. Recently, a few experiments have studied the self-cleavage activity of the HDV ribozyme during transcription (Chadalavada et al. 2000(Chadalavada et al. , 2002(Chadalavada et al. , 2007Diegelman-Parente and Bevilacqua 2002). A clear and detailed understanding of the kinetic process of RNA folding including the folding pathways during transcription is crucial for uncovering the mechanism of RNA functions. ...
... The cotranscriptional folding of the wild 99-nt HDV ribozyme can effectively avoid the meta-stable intermediate C7, which occupies ∼50% of the population and lasts ∼30 min and then transits to the native state in the refolding process (Chadalavada et al. 2000(Chadalavada et al. , 2002. The cotranscriptional folding also shows bifurcation folding behavior from step 34, one pathway as C2-C3-C5-C6-C8 would directly fold to the native state, another pathway folds to the intermediate state along C2-C4-C7. ...
Hepatitis delta virus (HDV) ribozyme performs the self-cleavage activity through folding to a double pseudoknot structure. The folding of functional RNA structures is often coupled with the transcription process. In this work, we developed a new approach for predicting the co-transcriptional folding kinetics of RNA secondary structures with pseudoknots. We theoretically studied the co-transcriptional folding behavior of the 99nt HDV sequence, two upstream flanking sequences and one downstream flanking sequence. During transcription, the 99nt HDV can effectively avoid the trap intermediates and quickly fold to the cleavage-active state. It is different from its refolding kinetics, which folds into an intermediate trap state. For all the sequences, the ribozyme regions (from 1 to 73) all fold to the same structure during transcription. However, the existence of the 30nt upstream flanking sequence can inhibit the ribozyme region folding into the active native state through forming an alternative helix Alt1 with the segment 70-90. The longer upstream flanking sequence 54nt itself forms a stable hairpin structure, that sequesters the formation of Alt1 helix and leads to rapid formation of cleavage-active structure. Although the 55nt downstream flanking sequence could invade the already folded active structure during transcription by forming a more stable helix with the ribozyme region, the slow transition rate could keep the structure in the cleavage-active structure to perform the activity.
... Since intermediate states of RNAs can be important to their biological functions [5,76,[80][81][82][83][84][85], unfolding pathway of RNAs including some pseudoknots has been studied through theoretical modeling and experiments [75][76][77][81][82][83][84][85][86][87][88]. To examine the unfolding pathway of RNA pseudoknots, we made comprehensive analyses for six RNA pseudoknots; see Fig 7 and S4 Fig. Based on the simulations for each pseudoknot at a given solution condition, beyond the fractions of states F and U, the fractions of different intermediate hairpin states (named as S1 and S2 for intermediate states reserving one of Stem 1 and Stem 2, respectively) at different temperatures can also be calculated; see Figs 7 and 8 and S4 and S6 Figs. ...
... Thus, with the increase of temperature, the dominating unfolding pathway of MMTV pseudoknot is F!S1!U overwhelming the pathway of F!S2!U; see unfolding processes almost undergo the only pathway F!S1!U through the intermediate state S1 and the other pathway of F!S2!U appears negligibly, as shown in S4 Fig. Interestingly, for T2 pseudoknot at 1M [K + ], there are two comparable unfolding pathways: F!S1!U and F!S2!U, where the population of S1 state is only slightly higher than that of S2 state; see Fig 7B. Our calculations are in accordance with the experiments [75][76][77][80][81][82][83] as well as the recent theoretical studies [59,[84][85][86][87][88]. The above analyses show that RNA pseudoknots of different sequences can have very different unfolding pathways. ...
RNA pseudoknots are a kind of minimal RNA tertiary structural motifs, and their three-dimensional (3D) structures and stability play essential roles in a variety of biological functions. Therefore, to predict 3D structures and stability of RNA pseudoknots is essential for understanding their functions. In the work, we employed our previously developed coarse-grained model with implicit salt to make extensive predictions and comprehensive analyses on the 3D structures and stability for RNA pseudoknots in monovalent/divalent ion solutions. The comparisons with available experimental data show that our model can successfully predict the 3D structures of RNA pseudoknots from their sequences, and can also make reliable predictions for the stability of RNA pseudoknots with different lengths and sequences over a wide range of monovalent/divalent ion concentrations. Furthermore, we made comprehensive analyses on the unfolding pathway for various RNA pseudoknots in ion solutions. Our analyses for extensive pseudokonts and the wide range of monovalent/divalent ion concentrations verify that the unfolding pathway of RNA pseudoknots is mainly dependent on the relative stability of unfolded intermediate states, and show that the unfolding pathway of RNA pseudoknots can be significantly modulated by their sequences and solution ion conditions.
... 122 Experimental and computational techniques can be used independently or in concert to further understand RNA folding landscapes. 80,190,191 The results in Chapter 2 outline a unified folding landscape ( Fig. 2 19,64,192,193 194,195,197 Their characterizations of upstream cleavage sequences with the ability to inhibit and accelerate cleavage, 195 as well as the effects of downstream sequences to foster alternative secondary structure motifs both within and outside of the ribozyme 194,197 further outlined various potential folding intermediates and kinetic traps within the HDV RNA in in-vivo-like conditions. These previous studies provided a broad-scale look at the folding free energy landscape of HDV RNA beyond the minimal ribozyme sequence. ...
... 122 Experimental and computational techniques can be used independently or in concert to further understand RNA folding landscapes. 80,190,191 The results in Chapter 2 outline a unified folding landscape ( Fig. 2 19,64,192,193 194,195,197 Their characterizations of upstream cleavage sequences with the ability to inhibit and accelerate cleavage, 195 as well as the effects of downstream sequences to foster alternative secondary structure motifs both within and outside of the ribozyme 194,197 further outlined various potential folding intermediates and kinetic traps within the HDV RNA in in-vivo-like conditions. These previous studies provided a broad-scale look at the folding free energy landscape of HDV RNA beyond the minimal ribozyme sequence. ...
Small catalytic RNAs (ribozymes) are a class of RNAs less than 200 nucleotides in length, which are able to catalyze both cleavage and subsequent re-ligation of their own phosphodiester backbones. Cleavage results in formation of 2???,3??? cyclic phosphate and 5??? hydroxyl termini. There are five naturally occurring members of this group: the hepatitis delta virus (HDV) ribozyme, the hairpin ribozyme, the hammerhead ribozyme, the Varkud Satellite (VS) ribozyme, and the glmS ribozyme. All of these RNAs employ a distinct active site fold and combination of mechanisms to perform their chemical function. Despite extensive biochemical and structural characterization, the structural requirements of catalysis for the HDV and hairpin ribozymes have not been ascertained. The work in this thesis interrogates the conformational dynamics of these ribozymes to provide deeper insight into their structure-function relationships. In Chapter 2, we utilize a combination of ensemble fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to outline a portion of the free energy folding pathway of the HDV ribozyme, rationalizing all extant crystal structures. Our MD simulations in Chapter 3 define specific structural roles for the two GU wobble pairs in the HDV ribozyme active site through mutational analysis. We show in Chapter 4, with the aid of newly developed constant pH MD simulations for nucleic acids, that experimental pKa measurements of an active site residue in the hairpin ribozyme reflect a minor conformational population. Taken together, the results in this thesis reveal the complex network of noncovalent interactions within the HDV and hairpin ribozymes that can have either favor or disfavor catalysis. Our results thus provide insights into the function of these two catalytic RNAs through interrogations of their structural dynamics. Such knowledge consequently provides a deeper understanding of catalytic RNAs, and all non-coding RNAs in general. A thorough knowledge of these complex biomolecules is of growing importance given the increasing recognition of the crucial roles played by RNA in all three domains of life.
... The most general conclusion from these studies was that RNA folding involves multiple steps, with highly structured intermediates being populated along the way. Since then, the presence of intermediates has been shown to be a general feature of RNA folding (Pan and Sosnick, 1997;Treiber et al., 1998;Pan and Woodson, 1998;Russell and Herschlag, 1999;Chadalavada et al., 2002;Chauhan and Woodson, 2008). The extensive formation of intermediates creates a significant challenge for studying RNA folding. ...
... It was used in early studies of ribosome assembly (Traub and Nomura, 1969;Held and Nomura, 1973), and since then has been applied to a broad range of RNAs [e.g. (Woodson and Cech, 1991;Emerick et al., 1996;Pan and Sosnick, 1997;Russell and Herschlag, 1999;Chadalavada et al., 2002;Su et al., 2003;Xiao et al., 2005;Brooks and Hampel, 2009)]. Catalytic activity has also been used to follow protein folding (Kiefhaber et al., 1990;Kiefhaber, 1995), but it is uniquely well-suited to RNA because the propensity of RNA to form kinetically-trapped intermediates causes folding of many RNAs to be slower than their catalytic reactions (Treiber and Williamson, 1999). ...
As RNAs fold to functional structures, they traverse complex energy landscapes that include many partially folded and misfolded intermediates. For structured RNAs that possess catalytic activity, this activity can provide a powerful means of monitoring folding that is complementary to biophysical approaches. RNA catalysis can be used to track accumulation of the native RNA specifically and quantitatively, readily distinguishing the native structure from intermediates that resemble it and may not be differentiated by other approaches. Here, we outline how to design and interpret experiments using catalytic activity to monitor RNA folding, and we summarize adaptations of the method that have been used to probe aspects of folding well beyond determination of the folding rates.
... The HDV ribozyme sequence was based on studies of fast-folding ribozyme presented previously ( [14][15][16]. In all experiments, two strands of RNA were used. ...
... The ribozyme strand used in this study was designed to crystallize readily for use in Raman crystallography experiments. The sequence is based on a fast-folding variant with less propensity to misfold than the native sequence (15,16). The substrate was provided to the ribozyme in trans and had a single nucleotide upstream of the cleavage site ( Figure 1A). ...
The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.
... The major limitation of the FLAP-based transcription assays is a relatively slow rate of the FLAP maturation that leads to the emergence of the fluorescence signal. Indeed, some RNA structures fold on the timescale of minutes and the folding kinetics is strongly affected by Mg 2+ concentration (Bhaskaran, Rodriguez-Hernandez, & Perona, 2012;Chadalavada, Senchak, & Bevilacqua, 2002;Chen, 2008). ...
Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.
... While the use of cis-acting ribozymes to control gene expression is a strategy that has been used by others (Prommana et al., 2013;Yen et al., 2004), to our knowledge this is the first study to use a ribozyme as a substitute for more traditional mRNA-destabilizing approaches with the purpose of producing a reporter mRNA with a shortened translational half-life. Selection of the HdV ribozyme in this study over the more often favoured hammerhead ribozyme was due to the former RNA exhibiting a slow rate of folding (Chadalavada et al., 2002). Both this feature, combined with the introduction of an intron within the HdV ribozyme so as to deposit an exon-junction complex on the ribozyme sequence, were expected to minimize mRNA cleavage prior to nuclear export. ...
Human rhinoviruses express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study we have compared both the combined and individual ability of the 2 proteases to shut down in cellulo gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different Human Rhinovirus (HRV) species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.
... Three misfolded states are observed in Figure 4C, labeled M 1 through M 3 , where designation as a misfold was based on high RMSD (>5Å), low native state structural content (NC), and high degrees of non-native structure (NNC), as is consistent with a recent study of Noe et al. (36). While RNA is known to be easily trapped in metastable misfolded structures (37) and misfolding has been predicted or observed for this and numerous other pseudoknots (38)(39)(40)(41), we emphasize that our normalization of the total number of non-native contacts present in a structure (using the largest total quantity of NNC observed) may underscore the overall degree of misfolding present for a given structural state. ...
Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 μs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 μs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.
... Due to its complexity, most studies of RNA tertiary folding have been descriptive. Studies to date have identified misfolded intermediates (29)(30)(31)(32), revealed multiple folding pathways (33,34) and functional states (35), and led to the general perspective that RNA folds on a rugged energetic landscape (18). Nevertheless, these conclusions drawn from qualitative studies have been limited and, even at times, misleading. ...
Significance
Many biological processes, including splicing, translation, and genome maintenance, require structured RNAs to fold into complex three-dimensional shapes. Our current understanding of these processes is based on distilling principles from descriptive folding studies. Moving toward predictive models will require coupling observed structural changes with kinetic and thermodynamic measurements. We have dissected P4-P6 RNA folding through distinct structural states and measured the rate and equilibrium constants for transitions between these states. Common kinetics found for RNA tertiary elements embedded in different structural contexts may help develop predictive folding models. Also, our results suggest that RNA folding may be well described by a model analogous to the diffusion-collision model for protein folding.
... On the other hand, the stability of local structure may amplify the challenge of finding the native structure rapidly [35,4,5]. Non-native second-ary structures can be long-lived, like their native counterparts [6][7][8][9], and even native contacts can slow folding if they form prematurely and must be disrupted to allow resolution of non-native structure elsewhere [10][11][12][13][14][15][16]. In light of these properties, it is perhaps not surprising that RNA folding is found experimentally to be rife with kinetically trapped folding intermediates [17,18]. ...
Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs≈0.6 min(-1), while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here, we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1 min(-1)). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the 'choice' is enforced by energy barriers that grow larger as folding progresses.
... Folding of RNA is a largely hierarchical process (i.e., secondary then tertiary structure formation) (Brion and Westhof 1997;Leontis et al. 2006;Greenleaf et al. 2008) because most tertiary structure assembles from preformed secondary structures. For example, tRNA folds through five intermediates involving combinations of four different helices prior to formation of tertiary structure (Riesner et al. 1973;Crothers et al. 1974), while the HDV ribozyme folds through numerous base-pairing states and misfolds (Isambert and Siggia 2000;Chadalavada et al. 2002;Brown et al. 2004). Some variations in overall RNA folding mechanisms have been reported, e.g., tertiary structure or proteins driving native secondary structure (Thirumalai 1998;Wu and Tinoco 1998;Duncan and Weeks 2010;Woodson 2010); however, even in these cases, native secondary structure typically precedes tertiary structure. ...
Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg(2+) ion concentrations are low, K(+) concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg(2+) (0.5-2 mM) and K(+) (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.
... 49,83 It has been appreciated for decades that RNAs are prone to misfolding, starting from pioneering studies with tRNA and 5S rRNA. 100,101 More recently, misfolding has been demonstrated for several group I introns (described further below), 102-107 the bacterial RNase P RNA, 108 and the hepatitis delta virus ribozyme, 109 leading to recognition of misfolding as a dominant theme in RNA folding. 91,110 A notable exception was thought to be the Azoarcus group I intron ribozyme, which is roughly half the length of the Tetrahymena ribozyme and forms tertiary structure within milliseconds. ...
DEAD-box proteins are superfamily 2 helicases that function in all aspects of RNA metabolism. They employ ATP binding and hydrolysis to generate tight, yet regulated RNA binding, which is used to unwind short RNA helices non-processively and promote structural transitions of RNA and RNA-protein substrates. In the last few years, substantial progress has been made toward a detailed, quantitative understanding of the structural and biochemical properties of DEAD-box proteins. Concurrently, progress has been made toward a physical understanding of the RNA rearrangements and folding steps that are accelerated by DEAD-box proteins in model systems. Here, we review the recent progress on both of these fronts, focusing on the mitochondrial DEAD-box proteins Mss116 and CYT-19 and their mechanisms in promoting the splicing of group I and group II introns.
... These data demonstrate the existence of a thermal activation barrier for folding and demonstrate that the assay is robust across a range of temperatures and folding rate constants-even though, at the higher temperature for this particular tRNA, the burst is not explicitly observed. The rate constant at 37°C is comparable to the unimolecular transition of a slow folding species in the small HDV ribozyme (0.1-0.2 min À1 ) (Chadalavada et al. 2002). ...
We describe a strategy for tracking Mg²⁺-initiated folding of ³²P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA₁(Gln) reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of ³²P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.
... These observations raise the possibility that syn nucleobases may render an RNA slow to fold or more prone to misfolds. Such observations have previously been made on pseudoknots, wherein intermediates can engage in alternative interactions that cannot form native tertiary structure (Chadalavada et al. 2002). ...
Biological RNAs, like their DNA counterparts, contain helical stretches, which have standard Watson-Crick base pairs in the anti conformation. Most functional RNAs also adopt geometries with far greater complexity such as bulges, loops, and multihelical junctions. Occasionally, nucleobases in these regions populate the syn conformation wherein the base resides close to or over the ribose sugar, which leads to a more compact state. The importance of the syn conformation to RNA function is largely unknown. In this study, we analyze 51 RNAs with tertiary structure, including aptamers, riboswitches, ribozymes, and ribosomal RNAs, for number, location, and properties of syn nucleobases. These RNAs represent the set of nonoverlapping, moderate- to high-resolution structures available at present. We find that syn nucleobases are much more common among purines than pyrimidines, and that they favor C2'-endo-like conformations especially among those nucleobases in the intermediate syn conformation. Strikingly, most syn nucleobases participate in tertiary stacking and base-pairing interactions: Inspection of RNA structures revealed that the majority of the syn nucleobases are in regions assigned to function, with many syn nucleobases interacting directly with a ligand or ribozyme active site. These observations suggest that judicious placement of conformationally restricted nucleotides biased into the syn conformation could enhance RNA folding and catalysis. Such changes could also be useful for locking RNAs into functionally competent folds for use in X-ray crystallography and NMR.
... It has long been recognized that formation of the P3–P7 structure is a rate-limiting step in refolding of the active ribozyme, and the slow kinetics can be converted by mutations destabilizing the alternative P3 base pairing or base triples, or by disconnecting the nonnative interactions (Pan and Woodson; 1998; Rook et al. 1998; Treiber et al. 1998; Ohki et al. 2001; Heilman- Miller and Woodson 2003a). Also, it has been reported that the refolding of the genomic HDV ribozyme is dominated by slow formation of the pseudoknot (Chadalavada et al. 2002). However, single molecule study has shown that the majority of Tetrahymena group I ribozyme molecules enter the nonproductive folding pathway (Zhuang et al. 2000), raising questions regarding the relevance of the slow P3–P7 ...
Pseudoknots play critical roles in packing the active structure of various functional RNAs. The importance of the P3-P7 pseudoknot in refolding of group I intron ribozymes has been recently appreciated, while little is known about the pseudoknot function in co-transcriptional folding. Here we used the Candida group I intron as a model to address the question. We show that co-transcriptional folding of the active self-splicing intron is twice as fast as refolding. The P3-P7 pseudoknot folds slowly during co-transcriptional folding at a rate constant similar to the folding of the active ribozyme, and folding of both P3-P7 and P1-P10 pseudoknots are inhibited by antisense oligonucleotides. We conclude that when RNA folding is coupled with transcription, formation of pseudoknot structures dominates the productive folding pathway and serves as a rate-limiting step in producing the self-splicing competent Candida intron.
... For the sake of experimental tractability, thermodynamic boxes are typically built up from two mutations to an RNA or DNA molecule of interest, and are thus often referred to as double mutant cycles (Fig. 13.2). If mutants are to be made to a larger RNA by a cloning method such as QuikChange (Stratagene), only two sets of mutant primers are needed, which provides experimental convenience (Chadalavada et al., 2002). A typical thermodynamic box consists of the following four sequences displayed at the corners of the box: A or B respectively, has been modified (Fig. 13.2). ...
Double and triple mutant thermodynamic cycles provide a means to dissect the cooperativity of RNA and DNA folding at both the secondary and tertiary structural levels through use of the thermodynamic box or cube. In this article, we describe three steps for applying thermodynamic cycles to nucleic acid folding, with considerations of both conceptual and experimental features. The first step is design of an appropriate system and development of hypotheses regarding which residues might interact. Next is implementing this design in terms of a tractable experimental strategy, with an emphasis on UV melting. The final step, and the one we emphasize the most, is interpreting mutant cycles in terms of coupling between specific residues in the RNA or DNA. Coupling free energy in the absence and presence of changes elsewhere in the molecule is discussed in terms of specific folding models, including stepwise folding and concerted changes. Last, we provide a practical section on the use of commercially available software (KaleidaGraph) to fit melting data, along with a consideration of error propagation. Along the way, specific examples are chosen from the literature to illustrate the methods. This article is intended to be accessible to the biochemist or biologist without extensive thermodynamics background.
... All transcripts contain a G11C mutation that biases the equilibrium between Alt P1 and P1 toward the native fold. 45 Transcripts were Bfa I run-offs that contain ribozyme and HDVderived RNA sequence only. RNA was transcribed, purified, and 5′-end-radiolabeled as described. ...
The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg(2+) ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg(2+)-independent and two Mg(2+)-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg(2+)-free channel is inverted relative to the fully Mg(2+)-dependent channel, with each having a near-neutral pKa. Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg(2+). However, subsequent studies on a double mutant (DM) ribozyme suggested that the pKa observed for WT in the absence of Mg(2+) arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg(2+). Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg(2+)-free pKa observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Brønsted base appears to be water or hydroxide ion depending on pH. The observed pKa's are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg(2+) being similar to that in the presence of Mg(2+) and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Brønsted base.
... A common practice to ensure conformational homogeneity is to heat-denature RNA samples and renature them over a shallow temperature gradient. While this method works well for crystallizing some RNAs, it is not eVective for others, such as the precursor HDV ribozyme, that tend to misfold [30,31]. Only 30-50% of freshly transcribed HDV ribozymes fold correctly and undergo self-cleavage, and heat renaturation of the inactive HDV ribozyme does not lead to signiWcantly more cleavage (A.K. and J.A.D., unpublished results). ...
Different complexes of ribosomal proteins with specific rRNA fragments have been crystallized and studied by our group during the last six years. There are several factors important for successful crystallization of RNA/protein complexes, among them: length and content of RNA fragments, homogeneity of RNA and protein preparations, stability of the complexes, conditions for mixing RNA and protein components before crystallization, effect of Se-Met on RNA/protein complex crystal quality. In this paper we describe findings and methodical details, which helped us to succeed in obtaining X-ray quality crystals of several RNA/protein complexes.
... The biological function of ribonucleic acids is intimately determined by the three-dimensional structure adopted, the RNA fold. 1 Especially for larger RNAs, the pathway to reach the native fold usually proceeds via intermediates that represent local minima in the RNA folding free energy landscape. 2,3 When these intermediates are separated by large energy barriers they constitute folding traps and, therefore, the timescale of the folding process may take up to minutes. 4,5 It is only recently that a single RNA sequence of about 150 nucleotides in length has been described to co-exist in two stable folds, each associated with a distinct ribozyme activity. ...
We investigate 25-34 nucleotide RNA sequences, that have been rationally designed to adopt two different secondary structures that are in thermodynamic equilibrium. Experimental evidence for the co-existence of the two conformers results from the NH...N 1H NMR spectra. When compared to the NH...N 1H NMR spectra of appropriate reference sequences the equilibrium position is easily quantifiable even without the assignment of the individual NH resonances. The reference sequences represent several Watson-Crick base-paired double helical segments, each encountered in either of the two conformers of the bistable target sequence. In addition, we rationalize the influence of nucleotide mutations on the equilibrium position of one of the bistable RNA sequences. The approach further allows a detailed thermodynamic analysis and the evaluation of secondary structure predictions for multistable RNAs obtained by computational methods.
The COVID-19 pandemic persists despite the development of effective vaccines. As such, it remains crucial to identify new targets for antiviral therapies. The causative virus of COVID-19, SARS-CoV-2, is a positive-sense RNA virus with RNA structures that could serve as therapeutic targets. One such RNA with established function is the frameshift stimulatory element (FSE), which promotes programmed ribosomal frameshifting. To accelerate identification of additional functional RNA elements, we introduce a novel computational approach termed the Functional RNA Identification (FRID) pipeline. The guiding principle of our pipeline, which uses established component programs as well as customized component programs, is that functional RNA elements have conserved secondary and pseudoknot structures that facilitate function. To assess the presence and conservation of putative functional RNA elements in SARS-CoV-2, we compared over 6,000 SARS-CoV-2 genomic isolates. We identified 22 functional RNA elements from the SARS-CoV-2 genome, 14 of which have conserved pseudoknots and serve as potential targets for small molecule or antisense oligonucleotide therapeutics. The FRID pipeline is general and can be applied to identify pseudoknotted RNAs for targeted therapeutics in genomes or transcriptomes from any virus or organism.
The ThiM riboswitch from E.coli is a typical mRNA device that modulates downstream gene expression by sensing TPP. The helix‐based RNA folding theory is used to investigate its detailed regulatory behaviors in cells. This RNA molecule is transcriptionally trapped in a state with the unstructured SD sequence in the absence of TPP, which induces downstream gene expression. As a key step to turn on gene expression, formation of this trapped state (the genetic ON state), highly depends on the co‐transcriptional folding of its wild‐type sequence. Instead of stabilities of the genetic ON and OFF states, the transcription rate, pause and ligand levels are combined to affect the ThiM riboswitch‐mediated gene regulation, which is consistent with a kinetic control model.
RNA pseudoknots are a kind of minimal RNA tertiary structural motifs, and their three-dimensional (3D) structures and stability play essential roles in a variety of biological functions. Therefore, to predict 3D structures and stability of RNA pseudoknots is essential for understanding their functions. In the work, we employed our previously developed coarse-grained model with implicit salt to make extensive predictions and comprehensive analyses on the 3D structures and stability for RNA pseudoknots in monovalent/divalent ion solutions. The comparisons with available experimental data show that our model can successfully predict the 3D structures of RNA pseudoknots from their sequences, and can also make reliable predictions for the stability of RNA pseudoknots with different lengths and sequences over a wide range of monovalent/divalent ion concentrations. Furthermore, we made comprehensive analyses on the unfolding pathway for various RNA pseudoknots in ion solutions. Our analyses for extensive pseudokonts and the wide range of monovalent/divalent ion concentrations verify that the unfolding pathway of RNA pseudoknots is mainly dependent on the relative stability of unfolded intermediate states, and show that the unfolding pathway of RNA pseudoknots can be significantly modulated by their sequences and solution ion conditions.
RNA folding has been studied extensively in vitro, typically under dilute solution conditions and abiologically high salt concentrations of 1 M Na⁺ or 10 mM Mg²⁺. The cellular environment is very different, with 20–40% crowding and only 10–40 mM Na⁺, 140 mM K⁺, and 0.5–2.0 mM Mg²⁺. As such, RNA structures and functions can be radically altered under cellular conditions. We previously reported that tRNAphe secondary and tertiary structures unfold together in a cooperative two-state fashion under crowded in vivo-like ionic conditions, but in a noncooperative multistate fashion under dilute in vitro ionic conditions unless in nonphysiologically high concentrations of Mg²⁺. The mechanistic basis behind these effects remains unclear, however. To address the mechanism that drives RNA folding cooperativity, we probe effects of cellular conditions on structures and stabilities of individual secondary structure fragments comprising the full-length RNA. We elucidate effects of a diverse set of crowders on tRNA secondary structural fragments and full-length tRNA at three levels: at the nucleotide level by temperature-dependent in-line probing, at the tertiary structure level by small-angle X-ray scattering, and at the global level by thermal denaturation. We conclude that cooperative RNA folding is induced by two overlapping mechanisms: increased stability and compaction of tertiary structure through effects of Mg²⁺, and decreased stability of certain secondary structure elements through the effects of molecular crowders. These findings reveal that despite having very different chemical makeups RNA and protein can both have weak secondary structures in vivo leading to cooperative folding.
RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.
The hepatitis delta virus (HDV) ribozyme, a small self-cleaving RNA originally identified in the human pathogen HDV, has been found to be broadly dispersed throughout life. In this article, we describe an integrated approach to understand the catalytic mechanism of this ribozyme that combines kinetics, crystallography, Raman spectroscopy, and calculations. Kinetics studies provide rate and binding parameters for protons and metal ions, and allow for design of properly folded and catalytically relevant RNAs for crystallography. Raman studies on these crystals provide direct evidence that the nucleobase of C75 has a shifted pK
a. Moreover, Raman crystallography and solution kinetics demonstrate that proton binding to the N3 of C75 couples anticooperatively with binding of a Mg2+ ion, suggesting that the two species are close in space. Extensive structural studies on this ribozyme suggest that the cleavage reaction proceeds through a combination of Lewis acid catalysis by a catalytic Mg2+ ion and general acid catalysis by the nucleobase of C75. Molecular dynamics and electrostatics calculations support the above mechanism and reveal an intensely electronegative pocket that plays key roles in positioning the catalytic metal ion and C75 for catalysis. Integrating the results of kinetics, X-ray crystallography, Raman crystallography, and molecular dynamics suggests that there is a second Mg2+ ion in the active site that is bound diffusely and may play a structural role. In sum, these four disparate approaches provide for a robust kinetic mechanism for the HDV ribozyme that lays groundwork for future studies into its detailed mechanism of dynamics and cleavage.
Gene mutations influence the folding kinetics of hepatitis delta virus (HDV) ribozyme. In this work, we study the effect of the double mutation on the folding kinetics of HDV ribozyme. By using the master equation method combined with RNA folding free energy landscape, we predict the folding kinetics of C13A:G82U and A16U:U79A mutated HDV sequences. Their folding pathways are identified by recursively searching the states with high net flux-in(out) population starting from the native state. The results indicate that the folding kinetics of C13A:G82U mutation sequence is bi-phasic, which is similar to the wild type (wtHDV) sequence. While the folding kinetics of A16U:U79A mutation sequence is mono-phasic, it quickly folds to the native state in 30 s. Thus, the folding kinetics of double mutated HDV ribozyme depends on the mutation sites. © 2015, Wuhan University and Springer-Verlag Berlin Heidelberg.
RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.
The hepatitis delta virus (HDV) ribozymes are catalytic RNAs capable of cleaving their own sugar-phosphate backbone. The HDV virus possesses the ribozymes in both sense and antisense genomic transcripts, where they are essential for processing during replication. These ribozymes have been the subject of intense biochemical scrutiny and have yielded a wealth of mechanistic insights. In recent years, many HDV-like ribozymes have been identified in nearly all branches of life. The ribozymes are implicated in a variety of biological events, including episodic memory in mammals and retrotransposition in many eukaryotes. Detailed analysis of additional HDV-like ribozyme isolates will likely reveal many more biological functions and provide information about the evolution of this unique RNA.
Double and triple mutant thermodynamic cycles provide a means to dissect the cooperativity of RNA and DNA folding at both the secondary and tertiary structural levels through use of the thermodynamic box or cube. In this article, we describe three steps for applying thermodynamic cycles to nucleic acid folding, with considerations of both conceptual and experimental features. The first step is design of an appropriate system and development of hypotheses regarding which residues might interact. Next is implementing this design in terms of a tractable experimental strategy, with an emphasis on UV melting. The final step, and the one we emphasize the most, is interpreting mutant cycles in terms of coupling between specific residues in the RNA or DNA. Coupling free energy in the absence and presence of changes elsewhere in the molecule is discussed in terms of specific folding models, including stepwise folding and concerted changes. Last, we provide a practical section on the use of commercially available software (KaleidaGraph) to fit melting data, along with a consideration of error propagation. Along the way, specific examples are chosen from the literature to illustrate the methods. This article is intended to be accessible to the biochemist or biologist without extensive thermodynamics background.
The self-cleaving RNA sequences, or ribozymes, in the genomic and antigenomic strands of hepatitis delta virus (HDV) RNA fold
into structures that are similar to each other but distinct from those of small ribozymes associated with the RNA replicons
that infect plants. HDV ribozymes have provided a tractable system for studying the mechanism of catalytic RNA, and results
of biochemical and structural studies on the HDV ribozymes, from a number of labs, have enhanced our understanding and expanded
our thinking about the potential for catalytic roles of RNA side chains. The results of these studies are consistent with
models suggesting that both an active-site cytosine and a divalent metal ion have catalytic roles in facilitating the cleavage
reaction in the HDV ribozymes. Despite recent advances, details about the catalytic mechanism of the HDV ribozyme continue
to be debated, and these ribozymes should serve as a good system for further study.
RNA pseudoknots are examples of minimal structural motifs in RNA with tertiary interactions that stabilize the structures of many ribozymes. They also play an essential role in a variety of biological functions that are modulated by their structure, stability, and dynamics. Therefore, understanding the global principles that determine the thermodynamics and folding pathways of RNA pseudoknots is an important problem in biology, both for elucidating the folding mechanisms of larger ribozymes as well as addressing issues of possible kinetic control of the biological functions of pseudoknots. We report on the folding/unfolding kinetics of a hairpin-type pseudoknot obtained with microsecond time-resolution in response to a laser temperature-jump perturbation. The kinetics are monitored using UV absorbance as well as fluorescence of extrinsically attached labels as spectroscopic probes of the transiently populated RNA conformations. We measure folding times of 1-6 ms at 37 °C, which are at least 100-fold faster than previous observations of very slow folding pseudoknots that were trapped in misfolded conformations. The measured relaxation times are remarkably similar to predictions of a computational study by Thirumalai and co-workers (Cho, S. S.; Pincus, D.L.; Thirumalai, D. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17349-17354). Thus, these studies provide the first observation of a fast-folding pseudoknot and present a benchmark against which computational models can be refined.
HDV ribozymes catalyze their own scission from the transcript during rolling circle replication of the hepatitis delta virus. In vitro selection of self-cleaving ribozymes from a human genomic library revealed an HDV-like ribozyme in the second intron of the human CPEB3 gene and recent results suggest that this RNA affects episodic memory performance. Bioinformatic searches based on the secondary structure of the HDV/CPEB3 fold yielded numerous functional ribozymes in a wide variety of organisms. Genomic mapping of these RNAs suggested several biological roles, one of which is the 5' processing of non-LTR retrotransposons. The family of HDV-like ribozymes thus continues to grow in numbers and biological importance.
RNAs and RNA-protein complexes (RNPs) traverse rugged energy landscapes as they fold to their native structures, and many continue to undergo conformational rearrangements as they function. Due to the inherent stability of local RNA structure, proteins are required to assist with RNA conformational transitions during initial folding and in exchange between functional structures. DEAD-box proteins are superfamily 2 RNA helicases that are ubiquitously involved in RNA-mediated processes. Some of these proteins use an ATP-dependent cycle of conformational changes to disrupt RNA structure nonprocessively, accelerating structural transitions of RNAs and RNPs in a manner that bears a strong resemblance to the activities of certain groups of protein chaperones. This review summarizes recent work using model substrates and tractable self-splicing intron RNAs, which has given new insights into how DEAD-box proteins promote RNA folding steps and conformational transitions, and it summarizes recent progress in identifying sites and mechanisms of DEAD-box protein activity within more complex cellular targets.
Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping
gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located
12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A.,
Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in
ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove
of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance
of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting
efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of
the triple helix in −1 frameshifting.
Kinetics and the atomic detail of RNA refolding are only poorly understood. It has been proposed that conformations with transient base pairing interaction are populated during RNA refolding, but a detailed description of those states is lacking. By NMR and CD spectroscopy, we examined the refolding of a bistable RNA and the influence of urea, Mg(2+), and spermidine on its refolding kinetics. The bistable RNA serves as a model system and exhibits two almost equally stable ground-state conformations. We designed a photolabile caged RNA to selectively stabilize one of the two ground-state conformations and trigger RNA refolding by in situ light irradiation in the NMR spectrometer. We can show that the refolding kinetics of the bistable RNA is modulated by urea, Mg(2+), and spermidine by different mechanisms. From a statistical analysis based on elementary rate constants, we deduce the required number of base pairs that need to be destabilized during the refolding transition and propose a model for the transition state of the folding reaction.
Self-cleaving RNAs have recently been identified in mammalian genomes. A small ribozyme related in structure to the hepatitis delta virus (HDV) ribozyme occurs in a number of mammals, including chimpanzees and humans, within an intron of the CPEB3 gene. The catalytic mechanisms for the CPEB3 and HDV ribozymes appear to be similar, generating cleavage products with 5'-hydroxyl and 2',3'-cyclic phosphate termini; nonetheless, the cleavage rate reported for the CPEB3 ribozyme is more than 6000-fold slower than for the fastest HDV ribozyme. Herein, we use full-length RNA and cotranscriptional self-cleavage assays to compare reaction rates among human CPEB3, chimp CPEB3, and HDV ribozymes. Our data reveal that a single base change of the upstream flanking sequence, which sequesters an intrinsically weak P1.1 pairing in a misfold, increases the rate of the wild-type human CPEB3 ribozyme by approximately 250-fold; thus, the human ribozyme is intrinsically fast-reacting. Secondary structure determination and native gel analyses reveal that the cleaved population of the CPEB3 ribozyme has a single, secondary structure that closely resembles the HDV ribozyme. In contrast, the precleavage population of the CPEB3 ribozyme appears to have a more diverse secondary structure, possibly reflecting misfolding with the upstream sequence and dynamics intrinsic to the ribozyme. Prior identification of expressed sequence tags (ESTs) in human cells indicated that cleavage activity of the human ribozyme is tissue-specific. It is therefore possible that cellular factors interact with regions upstream of the CPEB3 ribozyme to unmask its high intrinsic reactivity.
The full text of the dissertation is available as a Adobe Acrobat .pdf file (233 p.) ; Adobe Acrobat Reader required to view the file. Mode of access: World Wide Web. Thesis (Ph. D.)--Pennsylvania State University, 2004.
Self-cleaving hammerhead, hairpin, hepatitis delta virus, and glmS ribozymes comprise a family of small catalytic RNA motifs that catalyze the same reversible phosphodiester cleavage reaction, but each motif adopts a unique structure and displays a unique array of biochemical properties. Recent structural, biochemical, and biophysical studies of these self-cleaving RNAs have begun to reveal how active site nucleotides exploit general acid-base catalysis, electrostatic stabilization, substrate destabilization, and positioning and orientation to reduce the free energy barrier to catalysis. Insights into the variety of catalytic strategies available to these model RNA enzymes are likely to have important implications for understanding more complex RNA-catalyzed reactions fundamental to RNA processing and protein synthesis.
Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.
The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the double-rolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C(75)) with a pK(a) close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5'-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G(76) of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP(76) is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP(76) becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C(75) becomes positioned for reaction chemistry only along the trajectory from precursor to product.
Hepatitis delta virus (HDV) is a circular pathogenic RNA that uses self-cleavage by closely related 84 nt genomic and antigenomic ribozymes to facilitate the replication of its genome. Downstream of each ribozyme is a stretch of nucleotides termed the attenuator that functions to base-pair with and unfold the ribozyme into a rod-like fold. The competing rates of RNA synthesis, ribozyme folding and cleavage, and rod folding are therefore likely to affect the efficiency of co-transcriptional self-cleavage. In these studies, co-transcriptional folding of the genomic ribozyme was assayed in vitro by monitoring co-transcriptional self-cleavage of transcripts having variable lengths of sequence downstream of the ribozyme. Co-transcriptional cleavage data were simulated successfully only with kinetic models in which cleavage-inactive channels were populated during transcription. Partitioning to and escape from these channels was influenced, in part, by whether the available attenuator sequence could form structures with the ribozyme, and by the stability of such structures. Surprisingly, only 23 nt of attenuator were needed for strong inactivation of cleavage. Self-cleavage of certain 3'-virus-containing sequences could be restored, partially, by renaturation; however, self-cleavage of transcripts with a full-length attenuator could not be restored efficiently by renaturation in vitro. This suggests that in the presence of the attenuator, the cleavage-active ribozyme fold is not the thermodynamically most stable species. In accordance with this model, the efficiency of self-cleavage of the ribozyme followed by a full-length attenuator was increased by decreasing the rate of transcription. These results suggest that, in the absence of additional factors, efficient co-transcriptional cleavage of the full-length genomic HDV RNA may require cleavage to occur prior to synthesis of the attenuator.
In an effort to reduce the conformational heterogeneity of RNA, the modified nucleobase 8-bromoguanosine (8BrG) was introduced into oligonucleotides having the hairpin tetraloop motif YNMG (Y = U or C and M = C or A). Purine nucleobases with bromine at position eight are known to preferentially adopt the syn conformation as nucleosides. The hairpin tetraloop motif YNMG was chosen as a model system because it has a syn guanosine at position four of the loop that is essential for thermodynamic stability. Thermodynamic and structural characterization of modified oligonucleotides with the hairpin sequences UUCG, CGCG, and CGAG by UV-melting and NMR spectroscopy revealed that 8BrG substitution has a small effect upon the hairpin conformation, while the duplex conformation is strongly destabilized (DeltaDeltaG degrees 37 approximately +4.7 kcal mol-1), thus inhibiting dimerization. These results support a model in which 8BrG substitution shifts the hairpin-duplex equilibrium constant toward the hairpin conformation by destabilizing the duplex. This methodology should be useful for limiting conformational heterogeneity in large RNAs, with potential applications in structural biology and enzymology.
Prior studies of the metal ion dependence of the self-cleavage reaction of the HDV genomic ribozyme led to a mechanistic framework in which the ribozyme can self-cleave by multiple Mg2+ ion-independent and -dependent channels [Nakano et al. (2001) Biochemistry 40, 12022]. In particular, channel 2 involves cleavage in the presence of a structural Mg2+ ion without participation of a catalytic divalent metal ion, while channel 3 involves both structural and catalytic Mg2+ ions. In the present study, experiments were performed to probe the nature of the various divalent ion sites and any specificity for Mg2+. A series of alkaline earth metal ions was tested for the ability to catalyze self-cleavage of the ribozyme under conditions that favor either channel 2 or channel 3. Under conditions that populate primarily channel 3, nearly identical K(d)s were obtained for Mg2+, Ca2+, Ba2+, and Sr2+, with a slight discrimination against Ca2+. In contrast, under conditions that populate primarily channel 2, tighter binding was observed as ion size decreases. Moreover, [Co(NH3)6]3+ was found to be a strong competitive inhibitor of Mg2+ for channel 3 but not for channel 2. The thermal unfolding of the cleaved ribozyme was also examined, and two transitions were found. Urea-dependent studies gave m-values that allowed the lower temperature transition to be assigned to tertiary structure unfolding. The effects of high concentrations of Na+ on the melting temperature for RNA unfolding and the reaction rate revealed ion binding to the folded RNA, with significant competition of Na+ (Hill coefficient of 1.5-1.7) for a structural Mg2+ ion and an unusually high intrinsic affinity of the structural ion for the RNA. Taken together, these data support the existence of two different classes of metal ion sites on the ribozyme: a structural site that is inner sphere with a major electrostatic component and a preference for Mg2+, and a weak catalytic site that is outer sphere with little preference for a particular divalent ion.
Arylazide mediated photocrosslinking has been widely used to obtain structural constraints in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We establish a mechanistic framework for formation of adducts between photoactivated 3-hydroxyphenyl azide and RNA. Tethered to an internal site in an RNA duplex via a 2'-amido linkage, photolysis of the aryl azide yields a cross-strand cross-link. Analysis of the ability of reagents with diagnostic reactivities to intercept formation of this cross-strand cross-link supports the assignment that the photoactivated intermediate is the ketenimine or a ketenimine-derived ring expansion product. Neither the initially produced singlet nitrene nor the subsequently formed triplet nitrene contribute to cross-link formation. Argon matrix and time-resolved solution experiments show that photolysis of free 3-hydroxyphenyl azide releases (in <or=20 ns) either a ketenimine or azepinone intermediate that reacts with nucleophiles. Adenosine, uridine, and guanosine monophosphate nucleotides have approximately equivalent abilities to quench the cross-strand cross-link, indicating that the photoactivated intermediate reacts broadly with functional groups in RNA. The reactive intermediate forms an adduct with adenosine monophosphate when tethered to both an RNA duplex or unstructured single strand; thus, cross-link formation is independent of the local RNA environment. The lifetime of the reactive intermediate generated upon photolysis of free 3-hydroxyphenyl azide in 50 mM Hepes diamine buffer is found to be 60-160 micros or significantly shorter than large scale RNA folding events. RNA-tethered 3-hydroxyphenyl azide cross-linking in aqueous buffer can thus be used with confidence to map structural neighbors, including most dynamic interactions, in RNA.
The publishers wish to apologize for an error which occurred during printing of this article. Some text was inadvertantly
omitted from the legend to figure 1 during translation. The correct legend is published below.
The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.
Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical–accessible surface of an RNA to be mapped with
nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior
helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely
folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.
The sequence requirements for self-cleavage of hepatitis delta virus genomic RNA were examined using precursor RNAs which
were labeled at either the 5′ or 3′ ends and progressively deleted from the unlabeled end. In the presence of 50% formamide,
which enhances self-cleavage in 2 mM MgCI2 at 37°C, 84 nucleotides (nt) 3′ of the break site were required. In the absence of formamide the minimum was reduced to 82
nt. Under both sets of conditions, precursors with 1 nt 5′ to the break site cleaved. These results allowed two condition-dependent
minimal domains for self-cleavage to be defined. However, in the absence of formamide, sequences flanking the minimal domain
inhibited cleavage, possibly through Involvement in the formation of non-cleaving structures. These data are consistent with
the idea that cleavage in vivo could be regulated by alternative RNA structures.
Three models for the secondary structure of the hepatitis delta virus (HDV) antigenomic self-cleaving RNA element were tested
by site-directed mutagenesls. Two models In which bases 5' to the cleavage site are paired with sequence at the 3' end of
the element were both inconsistent with the data from the mutagenesis. Specifically, mutations in the 3' sequence which decrease
self-cleavage activity could not be compensated by base changes in the 5' sequence as predicted by these models. The evidence
was consistent with a third model In which the 3' end pairs with a portion of a loop within the ribozyme sequence to generate
a pseudoknot structure. This same pairing was also required to generate higher rates of cleavage In trans with a 15-mer ribozyme,
thus ruling out a proposed hammerhead-like 'axehead' model for the HDV ribozyme.
The Bacillus stearothermophilus ribosomal protein S15 binds to a phylogenetically conserved three-way junction formed by the intersection of helices 20, 21, and 22 of eubacterial 16S ribosomal RNA, inducing a large conformational change in the RNA. Like many RNA structures, this three-way junction can also be folded by the addition of polyvalent cations such as magnesium, as demonstrated by comparing the mobilities of the wild-type and mutant junctions in the absence and presence of polyvalent cations in nondenaturing polyacrylamide gels. Using a modification interference assay, critical nucleotides for folding have been identified as the phylogenetically conserved nucleotides in the three-way junction. NMR spectroscopy of the junction reveals that the conformations induced by the addition of magnesium or S15 are extremely similar. Thus, the folding of the junction is determined entirely by RNA elements within the phylogenetically conserved junction core, and the role of Mg2+ and S15 is to stabilize this intrinsically unstable structure. The organization of the junction by Mg2+ significantly enhances the bimolecular association rate (k(on)) of S15 binding, suggesting that S15 binds specifically to the folded form of the three-way junction via a tertiary structure capture mechanism.
In the ribozyme of hepatitis delta virus antigenomic RNA, two short duplexes, P2 and P2a, stabilize the active self-cleaving
structure. However, P2a also promotes kinetic trapping of non-native structures. A bulged adenosine (A14) separates P2a and
P2; this bulged A is conserved in clinical isolates of HDV but is unlikely to be physically close to the cleavage site phosphate
in the ribozyme structure. Removing the bulge did not significantly slow the rate of cleavage but slowed the conversion of
inactive to active conformations. In the absence of the bulged A, inactive conformations required higher urea concentrations
or higher temperatures to be activated. Thus, the bulged-nucleotide in the P2-P2a duplex did not provide an essential kink
or hinge between P2 and P2a that was required for cleavage activity but, rather, increased the rate of refolding from an inactive
to an active ribozyme structure. These data also suggest a model in which P2 and P2a form a coaxial stacked helix of 9 bp,
the most likely arrangement being one in which P2-P2a is roughly parallel to P1.
An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodynamic parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent experiments. Additional algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures? determined by comparative sequence analysis was assembled to allow optimization of parameters not based on experiments and to test the accuracy of the algorithm. On average, the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on average, has 86 % of known base-pairs. Experimental constraints, derived from enzymatic and flavin mononucleotide cleavage, improve the accuracy of structure predictions.
The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3' cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G.U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.
Ribozymes use a number of the same catalytic strategies as protein enzymes. However, general base catalysis by a ribozyme has not been demonstrated. In the hepatitis delta virus antigenomic ribozyme, imidazole buffer rescued activity of a mutant with a cytosine-76 (C76) to uracil substitution. In addition, a C76 to adenine substitution reduced the apparent pKa (where Ka is the acid constant) of the self-cleavage reaction by an amount consistent with differences in the pKa values of these two side chains. These results suggest that, in the wild-type ribozyme, C76 acts as a general base. This finding has implications for potential catalytic functions of conserved cytosines and adenines in other ribozymes and in ribonuclear proteins with enzymatic activity.
In the magnesium ion–dependent folding of theTetrahymena ribozyme, a kinetic intermediate accumulates in which the P4-P6 domain is formed, but the P3-P7 domain is not. The kinetic
barriers to P3-P7 formation were investigated with the use of in vitro selection to identify mutant RNA molecules in which
the folding rate of the P3-P7 domain was increased. The critical mutations disrupt native tertiary interactions within the
P4-P6 domain and increase the rate of P3-P7 formation by destabilizing a kinetically trapped intermediate. Hence, kinetic
traps stabilized by native interactions, and not simply by mispaired nonnative structures, can present a substantial barrier
to RNA folding.
Ribozymes use a number of the same catalytic strategies as protein enzymes. However, general base catalysis by a ribozyme
has not been demonstrated. In the hepatitis delta virus antigenomic ribozyme, imidazole buffer rescued activity of a mutant
with a cytosine-76 (C76) to uracil substitution. In addition, a C76 to adenine substitution reduced the apparent pK
a (whereK
a is the acid constant) of the self-cleavage reaction by an amount consistent with differences in the pK
a values of these two side chains. These results suggest that, in the wild-type ribozyme, C76 acts as a general base. This
finding has implications for potential catalytic functions of conserved cytosines and adenines in other ribozymes and in ribonuclear
proteins with enzymatic activity.
The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.
A model for the kinetic folding pathway of the Tetrahymena ribozyme has been proposed where the two main structural domains, P4-P6 and P3-P7, form in a hierarchical manner with P4-P6 forming first and P3-P7 folding on the minute timescale. Recent studies in our laboratory identified a set of mutations that accelerate P3-P7 formation, and all of these mutations appear to destabilize a native-like kinetic trap. To better understand the microscopic details of this slow step in the Tetrahymena ribozyme folding pathway, we have used a previously developed kinetic oligonucleotide hybridization assay to characterize the folding of several fast folding mutants. A comparison of the temperature dependence of P3-P7 folding between the mutant and wild-type ribozymes demonstrates that a majority of the mutations act by decreasing the activation enthalpy required to reach the transition state and supports the existence of the native-like kinetic trap. In several mutant ribozymes, P3-P7 folds with biphasic kinetics, indicating that only a subpopulation of molecules can evade the kinetic barrier. The rate of folding of the wild-type increases in the presence of urea, while for the mutants urea merely shifts the distribution between the two folding populations. Small structural changes or changes in solvent can accelerate folding, but these changes lead to complex folding behavior, and do not give rise to rapid two-state folding transitions. These results support the recent view of folding as an ensemble of molecules traversing a rugged energy landscape to reach the lowest energy state.
Large, structured RNAs traverse folding landscapes in which intermediates and long-lived misfolded states are common. To obtain a comprehensive description of the folding landscape for a structured RNA, it is necessary to understand the connections between productive folding pathways and pathways to these misfolded states. The Tetrahymena group I ribozyme partitions between folding to the native state and to a long-lived misfolded conformation. Here, we show that the observed rate constant for commitment to fold to the native or misfolded states is 1.9 min−1 (37 °C, 10 mM Mg2+), the same within error as the rate constant for overall folding to the native state. Thus, the commitment to alternative folding pathways is made late in the folding process, concomitant with or after the rate-limiting step for overall folding. The ribozyme forms much of its tertiary structure significantly faster than it reaches this commitment point and the tertiary structure is expected to be stable, suggesting that the commitment to fold along pathways to the native or misfolded states is made from a partially structured intermediate. These results allow the misfolded conformation to be incorporated into a folding framework that reconciles previous data and gives quantitative information about the energetic topology of the folding landscape for this RNA.
Hepatitis delta virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5'OH and 2',3'-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5' hydroxyl and 2', 3'- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.
The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.
In the magnesium ion-dependent folding of the Tetrahymena ribozyme, a kinetic intermediate accumulates in which the P4-P6 domain is formed, but the P3-P7 domain is not. The kinetic barriers to P3-P7 formation were investigated with the use of in vitro selection to identify mutant RNA molecules in which the folding rate of the P3-P7 domain was increased. The critical mutations disrupt native tertiary interactions within the P4-P6 domain and increase the rate of P3-P7 formation by destabilizing a kinetically trapped intermediate. Hence, kinetic traps stabilized by native interactions, and not simply by mispaired nonnative structures, can present a substantial barrier to RNA folding.
The folding kinetics of the catalytic domain of Bacillus subtilis ribonuclease P is analyzed here by fluorescence and catalytic activity. The folding pathway is apparently free of kinetic traps, as indicated by a decrease in folding rates upon the addition of urea. We apply Mg2+ and urea chevron analysis to fully describe the folding and unfolding kinetics of this ribozyme. A folding scheme containing two kinetic intermediates completely accounts for the free energy, the Mg2+ Hill coefficient and the surface buried in the equilibrium transition. At saturating Mg 2+concentrations, folding is limited by a barrier that is independent of Mg2+ and urea. These results describe the first trap-free folding pathway of a large ribozyme and indicate that kinetic traps are not an obligate feature of RNA folding.
A model for the secondary structure of the self-cleaving RNA from hepatitis delta virus was tested. Specific base changes were introduced in each of four regions with the potential for base-pairing (stems I-IV), and for each variant sequence, a rate constant for cleavage was determined. In each stem, mutations that would interfere with Watson-Crick base-pairing also reduced the first-order rate constants by 10-10(4)-fold relative to the unmodified version. Within stems I and II and a shortened form of stem IV, compensatory changes resulted in rates of cleavage equal to or greater than the unaltered ribozyme sequence. Stem III compensatory mutants cleaved faster than the uncompensated mutants although they were not as active as the natural sequence, suggesting additional sequence-dependent requirements within this region. Structure probing of RNA containing the stem II mutations provided an independent confirmation of stem II in the ribozyme. The predictive value of the model was tested by designing two trans-acting ribozymes which were circularly permuted composites of genomic, antigenomic, and unique sequences. The core of these two catalytic RNAs was the same, but they otherwise differed in that, in one of them, a constraining tetraloop sequence was added to stem II. Both ribozymes catalyzed the trans cleavage of a substrate oligoribonucleotide, thus providing additional evidence for stem II and the proposed structure in general.
Hepatitis delta virus genomic and antigenomic RNAs contain a self-cleavage site hypothesized to function in processing the viral RNA during replication. Self-cleavage requires only a divalent cation and is mediated at the genomic site by a sequence of less than 85 nucleotides. We propose that the genomic self-cleaving sequence element and a corresponding sequence from the anti-genomic RNA could generate related secondary structures. The region of the antigenomic sequence, predicted from the proposed structure, was synthesized and shown to be sufficient for self-cleavage. Evidence for two stems which form a tertiary interaction was obtained by site-specific mutagenesis of the antigenomic sequence. Efficient self-cleavage in 10 M formamide or 5 M urea, also a property of the genomic sequence, was dependent on base-pairing in both stems. But in the absence of denaturants, the stem distal to the site of cleavage was not required, suggesting that the tertiary interaction stabilizes the structure required for self-cleavage.
Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.
We report temperature-jump studies of the early melting transition of tRNAfMet (Escherichia coli). There are two measurable relaxation times τ, both independent of concentration and visible at 266 and 335 nm. The temperature dependence of the τ values establishes apparent activation energies, and implies that the process of structure formation has negligible activation energy, while dissociation of structure requires energy. We also measured the thermal difference spectrum for both relaxation effects, and found that each of these is apparently less G·C rich than the total tRNA melting. These results allow a general comparison of the rate of regenerating the "native" structure (zone I of the tRNA phase diagram), either from the low-salt (zone III) form, or from the zone II form (the product of the early melting transition). The conversion from the low-salt form is several orders of magnitude slower, and has a much larger activation energy. We conclude that zone II and zone III represent different conformations. Further consideration of all the factors leads to the conclusion that zone II is a structure with no noncloverleaf bonding, but in which the dihydrouridine helix of the cloverleaf may be melted. The kinetic results imply that the tertiary structure of tRNAfMet contains at least two regions of interaction whose melting is not obligatorily coupled. We present a simple hypothesis to interpret the results further, in which there are two interaction regions whose melting is virtually independent. With this hypothesis, we find that (in 0.17 M Na+) region 1 is formed in approximately 2-3 msec, and has a dissociation heat of about 22 kcal/mole, while region 2 is formed in about 7 msec, and has a dissociation heat of 51 kcal/mole. The melting of region 2 may include opening of the dihydrouridine helix, but all other interactions involve tertiary structure.
A sequence in the leader and first gene of the Escherichia coli alpha mRNA folds into a complex pseudoknot structure that is required for binding of a translational repressor. The thermal denaturation of a 112 nt RNA containing this structure has been followed by calorimetry and UV hyperchromicity. To determine the partially folded intermediates in unfolding, the denaturation of 13 mutants and of several fragments with successive deletions of helices were investigated as well. An unfolding pathway with seven states is proposed as the simplest mechanism that accounts for the data, and has several implications. (1) The lowest temperature transition appears only in the presence of moderate concentrations of Mg2+ or high concentrations of K+ (delta H approximately 45 kcal/mol), and is the unfolding of tertiary structures, rather than secondary structure. Under some conditions it is destabilized by increasing salt concentration. (2) Two of the intermediates unfolding at higher temperature must have non-canonical or tertiary interactions in addition to the known secondary structure. (3) Two alternative structures compete for formation of the complete pseudoknot, and form as the pseudoknot unfolds. Thus structures not present in the completely folded pseudoknot affect the overall thermodynamics, and probably the kinetics, of unfolding. (4) Approximately 16 kcal/mol of free energy is required to completely expose the coding region to ribosomes at 37 degrees C, though approximately 6.5 kcal/mol is regained by refolding of upstream regions after the pseudoknot is unfolded. The substantial energy needed to unfold the pseudoknot may affect the rate of translation from this ribosome binding site. A simple model of RNA folding in which an optimum secondary structure forms first, followed by tertiary interactions that further stabilize the secondary structure, does not hold in this RNA.
Hepatitis delta virus (HDV) contains a circular, viroid-like RNA genome, the only animal viral RNA of its kind. It possesses a ribozyme activity, which can autocatalytically cleave and ligate itself. The ribozyme has a unique structural requirement different from other known ribozymes. HDV RNA undergoes RNA-dependent RNA replication via a double rolling circle mechanism, which is probably mediated by cellular RNA polymerase II, utilizing modified cellular transcription machineries. HDV RNA encodes a single protein, hepatitis delta antigen, which is a nuclear, RNA-binding phosphoprotein and required for viral RNA replication. During replication, HDV RNA undergoes a specific RNA editing event to extend its open reading frame and produce a longer, isoprenylated delta antigen, which suppresses RNA replication and initiates viral particle assembly. Ribozyme, cell-mediated RNA-dependent RNA replication, and RNA editing are some of the unique properties and unresolved issues of the molecular biology of HDV.
We have investigated in detail the higher order structure of the genomic hepatitis delta virus (HDV) ribozyme using various base-specific chemical probes under native, semi-denaturing, and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethyl sulfate [which reacts with A (at N1) and C (at N3)] and a carbodiimide [which reacts with U (at N3) and G (at N1)]. In addition, for probing G residues (at N7), RNA samples were treated with NaBH4 and aniline after modification by treatment with dimethyl sulfate. The sites of modified positions were identified by primer extension analysis with reverse transcriptase. In general, our results are consistent with the proposed pseudoknot model of secondary structure, a model that is based on data from ribonucleolytic cleavage experiments. Our results provide clues to the identification of interacting bases in the HDV ribozyme. Furthermore, using this method we identified local conformational changes in several stem variants.
CBP2 is an RNA tertiary structure binding protein required for efficient splicing of a yeast mitochondrial group I intron. CBP2 must wait for folding of the two RNA domains that make up the catalytic core before it can bind. In a subsequent step, association of the 5' domain of the RNA is stabilized by additional interactions with the protein. Thus, CBP2 functions primarily to capture otherwise transient RNA tertiary structures. This simple one-RNA, one-protein system has revealed how the kinetic pathway of RNA folding can direct the assembly of a specific ribonucleoprotein complex. There are parallels to steps in the formation of a much more complex ribonucleoprotein, the 30S ribosomal subunit.
The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.
The sequence, secondary structure, and size requirements of the helix 2 region (H2) of a cis-acting hepatitis delta virus ribozyme Rz 1 were examined in this study. Mutational analysis was performed, and the cleavage rate of each H2 mutant of Rz 1 was assayed. We found that H2 could be elongated to twice its original size without affecting ribozyme folding while the shortening of H2 by one base pair severely decreased autolytic activity. In addition, the maintenance of the Watson-Crick base-pairing interactions of the last base pair of H2 (A16U58) was not critical for cis-cleavage reaction. Nevertheless, mutants with an AA, an AG, an AC, or a GG pair at the bottom of H2 were less active, and the sequence of the H2/H3 interface might affect the stability of the catalytic core. The negative effects on ribozyme folding, such as the destabilization of H2, the unfavorable sequences at the last base pair of H2 as well as the disruption of the continuity of H2 and H3, could be compensated for by elongating the H2 region of the corresponding mutants. The extension of H2 may alter the conformation of ribozyme molecules; in addition, it stabilized the catalytic core and enhanced the resistance to formamide. Finally, for a trans-acting ribozyme and its substrate that require the formation of H1, H2, and H4 to reconstitute the autocatalytic domain of HDV RNA, the extension of H2 stabilized the substrate/ribozyme complex and speeded up the cleavage rate but hindered the product release process.
Large ribozymes require divalent metal ions to fold. We show here that the tertiary structure of the Tetrahymena group I intron P4-P6 domain nucleates around a magnesium ion core. In the domain crystal structure, five magnesium ions bind in a three-helix junction at the centre of the molecule. Single atom changes in any one of four magnesium sites in this three-helix junction destroy folding of the entire 160-nucleotide P4-P6 domain. The magnesium ion core may be the RNA counterpart to the protein hydrophobic core, burying parts of the RNA molecule in the native structure.
A complex pseudoknot structure surrounds the first ribosome initiation site in the Escherichia coli alpha mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H+, K+ and Mg2+ concentrations. Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the "slow" form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form. A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C. Mg2+ and H+ appear to direct alpha mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K+ has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The alpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step.
The evidence showing that the self-assembly of complex RNAs occurs in discrete transitions, each relating to the folding of sub-systems of increasing size and complexity starting from a state with most of the secondary structure, is reviewed. The reciprocal influence of the concentration of magnesium ions and nucleotide mutations on tertiary structure is analyzed. Several observations demonstrate that detrimental mutations can be rescued by high magnesium concentrations, while stabilizing mutations lead to a lesser dependence on magnesium ion concentration. Recent data point to the central controlling and monitoring roles of RNA-binding proteins that can bind to the different folding stages, either before full establishment of the secondary structure or at the molten globule state before the cooperative transition to the final three-dimensional structure.
Hepatitis delta virus (HDV) is a small single-stranded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self-cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The self-cleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling-circle replication to unit-length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysis has not yet been proven. A minimal natural ribozyme sequence with proficient in vitro self-cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (P1-P4). The two pairings that define the 5' and 3' boundaries of the ribozyme, P1 and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo-cross-linking, mutagenesis and computer-assisted modeling provides clues to the three-dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5' end of P1, is in close proximity to three single-stranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans-acting ribozymes and smaller more active cis-acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.
The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.
The Tetrahymena thermophila self-splicing RNA is trapped in an inactive conformation during folding reactions at physiological temperatures. The structure of this metastable intermediate was probed by chemical modification interference and site-directed mutagenesis. In the inactive structure, an incorrect base-pairing, which we call Alt P3, displaces the P3 helix in the catalytic core of the intron. Mutations that stabilize Alt P3 increase the fraction of pre-rRNA that becomes trapped in the inactive structure, whereas mutations that destabilize Alt P3 reduce accumulation of this conformer. At high concentrations of Mg2+, the yield of correctly folded mutant pre-rRNAs is similar to wild-type RNA. Under these conditions, the rate of folding for mutant RNAs is slower than for the wild-type, but is increased by addition of urea. The results show that slow folding of the Tetrahymena pre-rRNA is a consequence of non-native secondary structure in the catalytic core of the intron, which is linked to an alternative hairpin in the 5' exon. This illustrates how kinetically stable, long-range interactions shape RNA folding pathways.
The secondary structure of the P5abc subdomain (a 56-nt RNA) of the Tetrahymena thermophila group I intron ribozyme has been determined by NMR. Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions. On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson-Crick base pairs, three G.U base pairs, and an extra-stable tetraloop are lost. The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct.
The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3 A resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5'-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.
The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.
The hammerhead ribozyme undergoes a well-defined two-stage folding process induced by the sequential binding of two magnesium ions. These probably correspond to the formation of domain 2 (0-500 microM magnesium ions) and domain 1 (1-20 mM magnesium ions), respectively. In this study we have used fluorescence resonance energy transfer (FRET) to analyze the ion-induced folding of a number of variants of the hammerhead ribozyme. We find that both A14G and G8U mutations are highly destabilizing, such that these species are essentially unfolded under all conditions. Thus they appear to be blocked in the first stage of the folding process, and using uranyl-induced photocleavage we show that the core is completely accessible to this probe under these conditions. Changes at G5 do not affect the first transition but appear to provide a blockage at the second stage of folding; this is true of changes in the sugar (removal of the 2'-hydroxyl group) and base (G5C mutation, previously studied by comparative gel electrophoresis). Arrest of folding at this intermediate stage leads to a pattern of uranyl-induced photocleavage that is changed from the wild-type, but suggests a structure less open than the A14G mutant. Specific photocleavage at G5 is found only in the wild-type sequence, suggesting that this ion-binding site is formed late in the folding process. In addition to folding that is blocked at selected stages, we have also observed misfolding. Thus the A13G mutation appears to result in the ion-induced formation of a novel tertiary structure.
Large ribozymes fold on a 'glacial' timescale compared to the folding of their protein counterparts. The sluggish folding exhibited by large RNAs results from the formation of kinetically trapped, misfolded intermediates, which are nonessential features of the folding mechanism. Newly developed mutant ribozymes that avoid kinetic traps should facilitate the study of the RNA folding problem.
The hepatitis D virus (HDV) relies on the helper hepatitis B virus (HBV) for the provision of its envelope, which consists of hepatitis B surface antigen (HBsAg). The RNA genome of HDV is a circular rod-like structure due to its extensive intramolecular base-pairing. HDV-RNA has ribozyme activity which includes autocatalytic cleavage and self-ligation properties, essential in virus replication via the rolling circle mechanism. Replication of the RNA is thought to be effected by cellular RNA polymerase II. Hepatitis D antigen (HDAg) is the only protein encoded by HDV-RNA and its long and short forms have a regulatory role in the replication and morphogenesis of the virus. Superinfected HBV carriers who become chronically infected with HDV are at increased risk of developing cirrhosis. Attempts to treat such carriers with interferon have not been particularly successful. In recent years the epidemiology of HDV has changed primarily due to the impact of HBV vaccination in preventing an increase in the pool of susceptible individuals. Copyright 1998 John Wiley & Sons, Ltd.
The structures of the model oligoribonucleotides that mimic the consecutive stages in the transcription of genomic HDV ribozyme have been analyzed by the Pb(2+)-induced cleavage method, partial digestion with specific nucleases and chemical probing. In the transcription intermediates, the P1 and P4 helical segments are found to be present in the final folded forms in which they exist in the full-length transcript. However, the region corresponding to the central hairpin forms another thermodynamically stable hairpin structure. Its correct folding requires the presence of a ribozyme 3'-terminal sequence and the formation of helix P2. This confirms the ribozyme structure of the pseudoknot type and points to the crucial role of helix P2 in its overall folding. Moreover, we show that the J4/2 region can be specifically cleaved in the presence of selected divalent metal ions in the full-length transcript, but not in a shorter one lacking six 3'-terminal nucleotides, which cannot form the pseudoknotted structure. Thus, a particular RNA conformation around that cleavage site is required for specific hydrolysis, and the J4/2 region seems to be involved in the formation of a general metal ion binding site. Recently, it has been proposed that, in the antigenomic ribozyme, a four nucleotide sequence within the J1/2 region may contribute to the folding pathway, being part of a mechanism responsible for controlling ribozyme cleavage activity. Our study shows that in the genomic ribozyme the central hairpin region may contribute to a similar mechanism, providing a barrier to the formation of an active structure in the ribozyme folding pathway.
The thermodynamic properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR experiments. The free energy increments at 37 degrees C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approximations are suggested for predicting stabilities of single mismatches in short RNA duplexes.
Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 A(2) of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85% of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free=27.9% for all reflections between 20.0 and 2.3 A) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification.
Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.
Many protein enzymes use general acid-base catalysis as a way to increase reaction rates. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH. The RNA enzyme (ribozyme) from hepatitis delta virus catalyzes self-cleavage of a phosphodiester bond. Reactivity-pH profiles in monovalent or divalent cations, as well as distance to the leaving-group oxygen, implicate cytosine 75 (C75) of the ribozyme as the general acid and ribozyme-bound hydrated metal hydroxide as the general base in the self-cleavage reaction. Moreover, C75 has a pK(a) perturbed to neutrality, making it "histidine-like." Anticooperative interaction is observed between protonated C75 and a metal ion, which serves to modulate the pK(a) of C75. General acid-base catalysis expands the catalytic repertoire of RNA and may provide improved rate acceleration.