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Riboswitches: The oldest mechanism for the regulation of gene expression?
Abstract
Riboswitches are structures that form in mRNA and regulate gene expression in bacteria. Unlike other known RNA regulatory structures, they are directly bound by small ligands. The mechanism by which gene expression is regulated involves the formation of alternative structures that, in the repressing conformation, cause premature termination of transcription or inhibition of translation initiation. Riboswitches regulate several metabolic pathways including the biosynthesis of vitamins (e.g. riboflavin, thiamin and cobalamin) and the metabolism of methionine, lysine and purines. Candidate riboswitches have also been observed in archaea and eukaryotes. The taxonomic diversity of genomes containing riboswitches and the diversity of molecular mechanisms of regulation, in addition to the fact that direct interaction of riboswitches with their effectors does not require additional factors, suggest that riboswitches represent one of the oldest regulatory systems.
... In other cases, such as the specific recognition and binding in the CRISPR-Cas9 gene editing system [23], RNA-DNA pairing is at the basis of the direct interaction with the DNA to be cut. Finally, both direct pairing and loop binding mechanisms are present in the regulatory RNAs called riboswitches, which upon binding to metabolites, control gene expression [24]. Riboswitches are aptamers characterized by structures made by few helix-loop units (see Fig. 1 and Sec. ...
... To a first approximation, then, stochastic effects that were neglected in writing Eqs. (23)(24)(25) can be captured by assuming that fluctuations around the minimum of L correspond to sampling molecular levels from a Boltzmann-Gibbs distribution ...
... (iv) Off-equilibrium properties, describing e.g. the approach to equilibrium or the transient response of the network to perturbations, can instead be derived by studying the linearized version of Eqs. (23)(24)(25) in the limit of small perturbations. [100] The above program has been carried out starting in 2013 over a series of papers by various authors, covering aspects ranging from the off-equilibrium dynamics of small miRNA-mediated circuits to the typical system-level properties of crosstalk in the human transcriptome (see Ref. [101] for a thorough review). ...
This contribution focuses on the fascinating RNA molecule, its sequence-dependent folding driven by base-pairing interactions, the interplay between these interactions and natural evolution, and its multiple regulatory roles. The four of us have dug into these topics using the tools and the spirit of the statistical physics of disordered systems, and in particular the concept of a disordered (energy/fitness) landscape. After an introduction to RNA molecules and the perspectives they open not only in evolutionary and synthetic biology but also in medicine, we will introduce the important notions of energy and fitness landscapes for these molecules. In Section III we will review some models and algorithms for RNA sequence-to-secondary-structure mapping. Section IV discusses how the secondary-structure energy landscape can be derived from unzipping data. Section V deals with the inference of RNA structure from evolutionary sequence data sampled in different organisms. This will shift the focus from the `sequence-to-structure' mapping described in Section III to a `sequence-to-function' landscape that can be inferred from laboratory evolutionary data on DNA aptamers. Finally, in Section VI, we shall discuss the rich theoretical picture linking networks of interacting RNA molecules to the organization of robust, systemic regulatory programs. Along this path, we will therefore explore phenomena across multiple scales in space, number of molecules and time, showing how the biological complexity of the RNA world can be captured by the unifying concepts of statistical physics.
... Ligandinduced alternative folding of the expression platform is exploited to regulate transcription, translation, or other gene expression processes ( Figure 1) [5][6][7][8][9][10]. Because all known riboswitch aptamers bind their target ligands without the need for protein factors, some of these RNAs might be representatives of an ancient sensory and regulatory system that was used by RNA World [11,12] organisms long before proteins emerged in evolution [1,[13][14][15]. ...
... Riboswitches as research tools Many riboswitch classes monitor or regulate fundamental biochemical pathways, and we and others have therefore concluded that they are likely to be of ancient origin [13][14][15]. Thus, each riboswitch offers researchers a simple mechanism to spy on one or more fundamental biological processes either to monitor normal physiological changes or to identify compounds that perturb cellular processes. ...
Riboswitches are structured noncoding RNA domains used by many bacteria to monitor the concentrations of target ligands and regulate gene expression accordingly. In the past 20 years over 55 distinct classes of natural riboswitches have been discovered that selectively sense small molecules or elemental ions, and thousands more are predicted to exist. Evidence suggests that some riboswitches might be direct descendants of the RNA-based sensors and switches that were likely present in ancient organisms before the evolutionary emergence of proteins. We provide an overview of the current state of riboswitch research, focusing primarily on the discovery of riboswitches, and speculate on the major challenges facing researchers in the field.
... The structural change results in upregulation or downregulation of expression of the protein(s) encoded in the same mRNA. 1,2,[4][5][6][7][8][9][10] It has been suggested that these riboswitches may be remnants of the ancient RNA world. 8,11 The possibility of chemical regulation of gene expression without direct involvement of proteins (e.g., transcription factors) has inspired many researchers to develop synthetic riboswitches that respond to non-natural molecules using RNA aptamers selected in vitro. ...
... 1,2,[4][5][6][7][8][9][10] It has been suggested that these riboswitches may be remnants of the ancient RNA world. 8,11 The possibility of chemical regulation of gene expression without direct involvement of proteins (e.g., transcription factors) has inspired many researchers to develop synthetic riboswitches that respond to non-natural molecules using RNA aptamers selected in vitro. While many synthetic riboswitches mimicking natural mechanisms have been designed in bacteria, synthetic riboswitches that function in other cells and organisms, and riboswitches based on mechanisms not found in nature have also been developed. ...
The emerging community of cell-free synthetic biology aspires to build complex biochemical and genetic systems with functions that mimic or even exceed those in living cells. To achieve such functions, cell-free systems must be able to sense and respond to the complex chemical signals within and outside the system. Cell-free riboswitches can detect chemical signals via RNA-ligand interaction and respond by regulating protein synthesis in cell-free protein synthesis systems. In this article, we review synthetic cell-free riboswitches that function in both prokaryotic and eukaryotic cell-free systems reported to date to provide a current perspective on the state of cell-free riboswitch technologies and their limitations.
... As these data are commensurate to met operon transcript stabilities, we conclude that varying mRNA degradation affects translation and the resulting protein amounts, with the Secondary structure determination revealed that the met leader RNA indeed harbors all structural features known from classical T-box riboswitches. By its size, however, the element stands out from the majority of other Tbox riboswitches, including those with methionyl-tRNA specificity (2,42). Interestingly, the met leader RNA Tbox riboswitch was previously shown to prefer interaction with initiator-methionyl-tRNA over elongator-methionyl-tRNA to activate the system (6). ...
... This is rather unusual as bacterial riboswitch turnover is commonly associated with the action of RNase P and RNase Y (36,37,60). The met leader terminator stem comprises a helix of 23 bp which is above the average length of terminator stems usually present in other T-box riboswitches (including MET-T-box systems) (2,42). RNase III requires double-stranded RNA stretches of 20-25 bp for cleavage, and it is therefore reasonable to suggest that the length of the stem in combination with presence of a GC-rich region renders the terminator an RNase III target (38,39). ...
In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.
... They usually impose a secondary-structural conformation on the mRNA to control translational efficiency of the transcript [73]. They have a widespread distribution of taxonomy and are capable of regulating highly conserved metabolic pathways, indicating their long history of gene regulatory mechanism [74]. To date, up to 50 classes of riboswitch are investigated, which involved in a variety of crucial biochemical pathways including co-enzymes, nucleobases, amino acids and single ions [74,75]. ...
... They have a widespread distribution of taxonomy and are capable of regulating highly conserved metabolic pathways, indicating their long history of gene regulatory mechanism [74]. To date, up to 50 classes of riboswitch are investigated, which involved in a variety of crucial biochemical pathways including co-enzymes, nucleobases, amino acids and single ions [74,75]. In cyanobacteria, only a few riboswitches are studied for their application in regulation of gene expression. ...
Cyanobacteria are Gram-negative photoautotrophic prokaryotes and have shown great importance to the Earth’s ecology. Based on their capability in oxygenic photosynthesis and genetic merits, they can be engineered as microbial chassis for direct conversion of carbon dioxide to value-added biofuels and chemicals. In the last decades, attempts have given to the application of synthetic biology tools and approaches in the development of cyanobacterial cell factories. Despite the successful proof-of-principle studies, large-scale application is still a technical challenge due to low yields of bioproducts. Therefore, recent efforts are underway to characterize and develop genetic regulatory parts and strategies for the synthetic biology applications in cyanobacteria. In this review, we present the recent advancements and application in cyanobacterial synthetic biology toolboxes. We also discuss the limitations and future perspectives for using such novel tools in cyanobacterial biotechnology.
... R iboswitches are cis-acting regulatory mRNA elements that are usually located in the 5′ untranslated region (5′UTR) of a messenger RNA (mRNA) and control gene expression by directly sensing small molecules [1][2][3][4][5] . Since their first discovery in 2002 1,6,7 , riboswitches have become recognized as important and widespread regulators of genes involved in many bacterial cellular processes [8][9][10][11][12] . ...
... This system of regulation in E. coli appears to be conserved in a high proportion of Gram-negative bacteria including the Xanthomonas genus 24 . Although potential riboswitches involved in the regulation of Met biosynthesis genes have been proposed in Gram-negative bacteria 3,24 , none of them has been functionally characterized. T-box riboswitches have long been thought to exist primarily in Gram-positive bacteria 8,11,12,[19][20][21][22]25 . ...
All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species. Riboswitches consist of an aptamer domain and expression platform, which senses a signal and regulates gene expression, respectively. Here the authors show that the expression platform of a SAM-I riboswitch from the Gram-negative bacteria can sense and bind uncharged an initiator Met tRNA to control the met operon.
... The means by which the switching mechanism mediates gene regulation is diverse, and includes transcription attenuation, translation initiation, dual control of transcription and translation, control of RNA stability, and RNA splicing. [1][2][3][4] For some riboswitches, a common, conserved ligand-binding aptamer may be coupled to different expression platforms and thereby regulate gene expression through distinct mechanisms (e.g., transcription or translation) in different bacterial species. [5][6][7] In all cases, however, the fate of gene expression is tightly controlled through ligand-induced conformational changes in the aptamer domain that are transmitted to the expression platform. ...
... Furthermore, several purine-derived molecules are used by bacteria as signaling molecules that are considered to have RNA World origins (51,52). Some riboswitches have also been proposed to be descendant from the major sensory and regulatory components used by ancient organisms long before proteins emerged as dominant players in evolution (1,4,5,53). If true, then the discovery and analysis of purine-sensing riboswitches might permit us to infer the structures and functions of riboswitches from some of the earliest forms of life. ...
Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2′-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic “ON” switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.
... The means by which the switching mechanism mediates gene regulation is diverse, and includes transcription attenuation, translation initiation, dual control of transcription and translation, control of RNA stability, and RNA splicing. [1][2][3][4] For some riboswitches, a common, conserved ligand-binding aptamer may be coupled to different expression platforms and thereby regulate gene expression through distinct mechanisms (e.g., transcription or translation) in different bacterial species. [5][6][7] In all cases, however, the fate of gene expression is tightly controlled through ligand-induced conformational changes in the aptamer domain that are transmitted to the expression platform. ...
The thiamine pyrophosphate (TPP)-sensing riboswitch is one of the earliest discovered and most widespread riboswitches. Numerous structural studies have been reported for this riboswitch bound with various ligands. However, the ligand-free (apo) structure remains unknown. Here, we report a 3.1 Å resolution crystal structure of Escherichia coli TPP riboswitch in the apo state, which exhibits an extended, Y-shaped conformation further supported by small-angle X-ray scattering data and driven molecular dynamics simulations. The loss of ligand interactions results in helical uncoiling of P5 and disruption of the key tertiary interaction between the sensory domains. Opening of the aptamer propagates to the gene-regulatory P1 helix and generates the key conformational flexibility needed for the switching behavior. Much of the ligand-binding site at the three-way junction is unaltered, thereby maintaining a partially preformed pocket. Together, these results paint a dynamic picture of the ligand-induced conformational changes in TPP riboswitches that confer conditional gene regulation.
... Some species cluster all the RBP genes into a single operon, whereas other species disperse the RBP genes along the chromosome in various transcriptional units [11]. The expression of RBP and transporter genes may be regulated by the FMN riboswitch, a genetic element found upstream of several rib operons and monocistronic rib genes [18,[23][24][25][26][27]. FMN binds to the aptamer portion of the FMN riboswitch, inhibiting the transcription and or translation of the downstream genes [2,23]. ...
Active flavins derived from riboflavin (vitamin B2) are essential for life. Bacteria biosynthesize riboflavin or scavenge it through uptake systems, and both mechanisms may be present. Because of riboflavin's critical importance, the redundancy of riboflavin biosynthetic pathway (RBP) genes might be present. Aeromonas salmonicida, the aetiological agent of furunculosis, is a pathogen of freshwater and marine fish, and its riboflavin pathways have not been studied. This study characterized the A. salmonicida riboflavin provision pathways. Homology search and transcriptional orchestration analysis showed that A. salmonicida has a main riboflavin biosynthetic operon that includes ribD, ribE1, ribBA, and ribH genes. Outside the main operon, putative duplicated genes ribA, ribB and ribE, and a ribN riboflavin importer encoding gene, were found. Monocistronic mRNA ribA, ribB and ribE2 encode for their corresponding functional riboflavin biosynthetic enzyme. While the product of ribBA conserved the RibB function, it lacked the RibA function. Likewise, ribN encodes a functional riboflavin importer. Transcriptomics analysis indicated that external riboflavin affected the expression of a relatively small number of genes, including a few involved in iron metabolism. ribB was downregulated in response to external riboflavin, suggesting negative feedback. Deletion of ribA, ribB and ribE1 showed that these genes are required for A. salmonicida riboflavin biosynthesis and virulence in Atlantic lumpfish (Cyclopterus lumpus). A. salmonicida riboflavin auxotrophic attenuated mutants conferred low protection to lumpfish against virulent A. salmonicida. Overall, A. salmonicida has multiple riboflavin endowment forms, and duplicated riboflavin provision genes are critical for A. salmonicida infection.
... For instance, the catalytic function of ribozymes can often be analyzed in terms of basic structural motifs, such as hammerhead or hairpin structures [2]. Other RNAs, like riboswitches, involve changes between alternative structures [3]. Understanding the relation sequence and structure is therefore a central challenge in molecular biology. ...
We propose a novel heuristic to predict RNA secondary structure formation pathways that has two components: (i) a folding algorithm and (ii) a kinetic ansatz. This heuristic is inspired by the kinetic partitioning mechanism, by which molecules follow alternative folding pathways to their native structure, some much faster than others. Similarly, our algorithm RAFFT starts by generating an ensemble of concurrent folding pathways ending in multiple metastable structures, which is in contrast with traditional thermodynamic approaches that find single structures with minimal free energies. When we constrained the algorithm to predict only 50 structures per sequence, near-native structures were found for RNA molecules of length ≤ 200 nucleotides. Our heuristic has been tested on the coronavirus frameshifting stimulation element (CFSE): an ensemble of 68 distinct structures allowed us to produce complete folding kinetic trajectories, whereas known methods require evaluating millions of sub-optimal structures to achieve this result. Thanks to the fast Fourier transform on which RAFFT (RNA folding Algorithm wih Fast Fourier Transform) is based, these computations are efficient, with complexity O ( L 2 log L ) .
... B. apis A29 appears to be shunting its metabolic energies into production of the essential amino acid lysine, which may be particularly valuable to developing larvae. Lysine appears to play an important role in other holometabolous symbioses; bacterial lysine synthesis and export is crucial for whitefly reproduction, and lysine synthesis is maintained in two Campotonus ant symbionts despite genome-wide erosion of central metabolic genes [70,77,78]. Indeed, our finding that cationic amino acid permeases have been gained and maintained suggests that in the evolution of Bombella in honey bee association, the transport of amino acids, such as lysine, was an important trait. ...
Honey bees have suffered dramatic losses in recent years, largely due to multiple stressors underpinned by poor nutrition [1]. Nutritional stress especially harms larvae, who mature into workers unable to meet the needs of their colony [2]. In this study, we characterize the metabolic capabilities of a honey bee larvae-associated bacterium, Bombella apis (formerly Parasaccharibacter apium), and its effects on the nutritional resilience of larvae. We found that B. apis is the only bacterium associated with larvae that can withstand the antimicrobial larval diet. Further, we found that B. apis can synthesize all essential amino acids and significantly alters the amino acid content of synthetic larval diet, largely by supplying the essential amino acid lysine. Analyses of gene gain/loss across the phylogeny suggest that four amino acid transporters were gained in recent B. apis ancestors. In addition, the transporter LysE is conserved across all sequenced strains of B. apis. Finally, we tested the impact of B. apis on developing honey bee larvae subjected to nutritional stress and found that larvae supplemented with B. apis are bolstered against mass reduction despite limited nutrition. Together, these data suggest a novel role of B. apis as a nutritional mutualist of honey bee larvae.
... descendants from molecular sensors that existed long before proteins began to predominate as functional polymers (12,23,24). ...
Significance
Numerous purines and their metabolic derivatives must be monitored for proper control of relevant metabolic pathways. In certain bacteria, some metabolic steps related to purine production or degradation are catalyzed by proteins whose production is under direct regulatory control by purine-sensing riboswitches. Four riboswitch classes selective for guanine, adenine, and 2′-deoxyguanosine (two classes) reported previously exploit a common architecture involving a three-stem junction. Here, we describe three additional classes based on this same scaffold that sense xanthine, guanine, or 2′-deoxyguanosine. Thus, some riboswitches can diversify their ligand-sensing and gene-control functions without the need to evolve entirely novel structures, which highlights a capability that could have also been exploited by ancient forms of life during the RNA World.
... 10 Numerous excellent reviews have been published recently on this fascinating subject. [11][12][13][14][15][16][17][18] A recent article hypothesized that an RNAT (RNA thermometer) also regulates a bacterial gene encoding a member of a cation diffusion facilitator family. Regulation of temperature thus provides a mechanism to overcome innate immune system mediated Zn(II) toxicity. ...
Mutation induced thermosensing ability of MicroROSE thermometer.
... As an alternative strategy to enable cell-free systems to respond to chemical signals, riboswitches have attracted increasing attention. Mainly found in bacteria, riboswitches are regulatory RNA sequences located in the untranslated regions (UTRs) of mRNAs that can upregulate or downregulate gene expression in response to specific chemical signals without protein factors (20)(21)(22)(23)(24)(25). A canonical riboswitch contains an aptamer domain that specifically recognizes a ligand molecule, and an expression platform that mediates a structural change upon aptamer-ligand interaction resulting in modulation of protein expression (26). ...
Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.
... The TPP-binding riboswitches are splicing regulators and responsible for mRNA stability and controlling the synthesis of thiamine in E. coli (Cheah et al., 2007) as depicted in Figure 7. The presence of TPP-binding riboswitches in eukaryotes provides evidence that riboswitches evolutionarily originate from a common ancestor of prokaryotic and eukaryotic organisms (Vitreschak et al., 2004). ...
Survival of microorganisms depends to a large extent on environmental conditions and the occupied host. By adopting specific strategies, microorganisms can thrive in the surrounding environment and, at the same time, preserve their viability. Evading the host defenses requires several mechanisms compatible with the host survival which include the production of RNA thermometers to regulate the expression of genes responsible for heat or cold shock as well as of those involved in virulence. Microorganisms have developed a variety of molecules in response to the environmental changes in temperature and even more specifically to the host they invade. Among all, RNA‐based regulatory mechanisms are the most common ones, highlighting the importance of such molecules in gene expression control and novel drug development by suitable structure‐based alterations.
This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
RNA in Disease and Development > RNA in Disease
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
... The adaptors, the receptors, are now proteins in nature, but sensing can also be achieved by RNAs (Frommer et al., 2015;Winkler and Breaker, 2003). Riboswitches (Vitreschak et al., 2004) are case in points. They have different conformations based on the environment, i.e. the presence of an effector. ...
Major evolutionary transitions as well as the evolution of codes of life are key elements in macroevolution which are characterized by increase in complexity Major evolutionary transitions ensues by a transition in individuality and by the evolution of a novel mode of using, transmitting or storing information. Here is where codes of life enter the picture: they are arbitrary mappings between different (mostly) molecular species. This flexibility allows information to be employed in a variety of ways, which can fuel evolutionary innovation. The collation of the list of major evolutionary transitions and the list of codes of life show a clear pattern: codes evolved prior to a major evolutionary transition and then played roles in the transition and/or in the transformation of the new individual. The evolution of a new code of life is in itself not a major evolutionary transition but allow major evolutionary transitions to happen. This could help us to identify new organic codes.
... hammerhead or hairpin structures (2). Other RNAs, like riboswitches, involve changes between alternative structures (3). Understanding the relation sequence and structure is therefore a central challenge in molecular biology. ...
We propose a novel heuristic to predict RNA secondary structures. The algorithm is inspired by the kinetic partitioning mechanism, by which molecules follow alternative folding pathways to their native structure, some much faster than others. Similarly, our algorithm RAFFT generates an ensemble of concurrent folding pathways ending in multiple metastable structures for each given sequence; this is in contrast with traditional thermodynamic approaches, which are based on aim to find single structures with minimal free energies. When analyzing 50 predicted folds per sequence, we found near-native predictions (79% PPV and 81% sensitivity) for RNAs of length < 200 nucleotides, matching the performance of recent deep-learning-based structure prediction methods. Our algorithm also acts as a folding kinetic ansatz, which we tested on two RNAs: the coronavirus frameshifting stimulation element (CFSE) and a classic bi-stable sequence. For the CFSE, an ensemble of 68 distinct structures computed by RAFFT allowed us to produce complete folding kinetic trajectories, whereas known methods require evaluating millions of sub-optimal structures to achieve this result. For the second application, only 46 distinct structures were required to reproduce the kinetics, whereas known methods required a sample of 20,000 structures. Thanks to the efficiency of the fast Fourier transform on which RAFFT is based, these computations are efficient, with complexity O(L^2 log L).
... A focus of our research team in recent years has been the discovery of riboswitches that sense specific metabolites or inorganic ions to regulate the expression of genes whose protein products synthesize, transport, utilize, or respond to the ligands being monitored. Over 50 distinct classes of riboswitches have been experimentally validated to date [11][12][13][14][15][16][17][18][19][20][21][22][23], and many of these sense nucleotide-derived compounds proposed to have been present during an era of biology before the evolutionary emergence of proteins [24][25][26][27][28]. Thus, uncovering additional classes of riboswitches promises to reveal more about how modern cells regulate critical biochemical processes, and perhaps also to provide insight into the functions of ancient RNA-based regulatory systems [29,30]. ...
Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding ‘intergenic regions’ (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel ribo-switch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.
... Riboswitches allosterically regulate gene expression through binding of a small-molecule ligand to the aptamer domain causing a conformational change, which then results in modification of specific gene expression [2]. This can for example contribute to regulation of activity of metabolic pathways such as the biosynthesis of vitamins (e.g., riboflavin, thiamine and cobalamin) and the metabolism of methionine, L-lysine and purines [3]. The mechanisms of action of riboswitches frequently found in bacterial species include transcription termination, translation initiation, mRNA degradation, splicing or RNA interference, with transcription termination being the most frequent [4,5]. ...
Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5′ untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.
... Interestingly, most of the ~50 known classes of riboswitches found in modern bacteria selectively sense and respond to RNA building blocks or their precursors, nucleotide-like coenzymes, and signaling molecules derived from RNA nucleotides 16,17 (Fig. 1B). Given their ligand specificities, abundances, and phylogenetic distributions, it has been proposed that some of these riboswitch classes might have originated during the RNA World 18,19 . Indeed, some riboswitches might be direct descendants from analogous ribozymes that used these ligands as coenzymes or substrates in ancient chemical reactions 20 , or as allosteric effectors. ...
The RNA World theory encompasses the hypothesis that sophisticated ribozymes and riboswitches were the primary drivers of metabolic processes in ancient organisms. Several types of catalytic RNAs and many classes of ligand-sensing RNA switches still exist in modern cells. Curiously, allosteric ribozymes formed by the merger of RNA enzyme and RNA switch components are largely absent in today’s biological systems. This is true despite the striking abundances of various classes of both self-cleaving ribozymes and riboswitch aptamers. Here we present the known types of ligand-controlled ribozymes and riboswitches and discuss the possible reasons why fused ribozyme–aptamer constructs have been disfavored through evolution. This Perspective summarizes the known types of ligand-controlled ribozymes and riboswitches and discusses the reasons why allosteric ribozymes formed by fusion of RNA enzymes and RNA aptamers are rare in today’s biological systems.
... In some cases, proteins with alternative primary functions have been shown to be direct regulators of their own translation [38][39][40][41][42] . Such examples highlight the enormous functional capacity of RNA where it can become a sensor of the cellular environment and autonomously regulate its own fate, as exemplified in cases where no protein is required, such as riboswitches [43][44][45][46][47] . When the effector being recognized by the mRNA is its own gene product, the result is autoregulation of gene expression. ...
Post-transcriptional autoregulation of gene expression is common in bacteria but many fewer examples are known in eukaryotes. We used the yeast collection of genes fused to GFP as a rapid screen for examples of feedback regulation in ribosomal proteins by overexpressing a non-regulatable version of a gene and observing the effects on the expression of the GFP-fused version. We tested 95 ribosomal protein genes and found a wide continuum of effects, with 30% showing at least a 3-fold reduction in expression. Two genes, RPS22B and RPL1B, showed over a 10-fold repression. In both cases the cis-regulatory segment resides in the 5’ UTR of the gene as shown by placing that segment of the mRNA upstream of GFP alone and demonstrating it is sufficient to cause repression of GFP when the protein is over-expressed. Further analyses showed that the intron in the 5’ UTR of RPS22B is required for regulation, presumably because the protein inhibits splicing that is necessary for translation. The 5’ UTR of RPL1B contains a sequence and structure motif that is conserved in the binding sites of Rpl1 orthologs from bacteria to mammals, and mutations within the motif eliminate repression.
... Most diverse is the distribution of THI-riboswitch like elements that has been witnessed in eubacteria, archaea and eukaryotes. Subsequent feature is that riboswitch like elements regulate a number of different processes; the most characteristic situation is the standard attenuation mechanism (Vitreschak et al., 2004). ...
... In some cases, proteins with alternative primary functions have been shown to be direct regulators of their own translation [38][39][40][41][42] . Such examples highlight the enormous functional capacity of RNA where it can become a sensor of the cellular environment and autonomously regulate its own fate, as exemplified in cases where no protein is required, such as riboswitches [43][44][45][46][47] . When the effector being recognized by the mRNA is its own gene product, the result is autoregulation of gene expression. ...
Post-transcriptional autoregulation of gene expression is common in bacterial systems but many fewer examples are known in eukaryotes. We used the yeast collection of genes fused to GFP as a rapid screen for examples of feedback regulation in ribosomal proteins by overexpressing a non-regulatable version of a gene and observing the effects on the expression of the GFP-fused version. We tested 95 ribosomal protein genes and found that 21 of them showed at least a three-fold repression. Two genes, RPS22B and RPL1B, showed over a 10-fold repression. In both cases the cis-regulatory segment resides in the 5-prime UTR of the gene as shown by placing that segment of the mRNA upstream of GFP alone and demonstrating it is sufficient to cause repression of GFP when the protein is over-expressed. Further analyses showed that the intron in the 5-prime UTR of RPS22B is required for regulation, presumably because the protein inhibits splicing that is necessary for translation. The 5-prime UTR of RPL1B contains a sequence and structure motif that is conserved in the binding sites of Rpl1 orthologs from bacteria to mammals, and mutations within the motif eliminate repression.
... Bioinformatic analyses have suggested that in some non-Firmicutes, S-box riboswitches regulate gene expression by mechanisms other than transcription attenuation, based on the absence of a distinct terminator helix in the expression platform (16)(17)(18). We used the Rfam database (19) to identify S-box sequences that were likely to regulate gene expression at the level of translation initiation, and for which the riboswitch of the equivalent gene has been characterized in B. subtilis. ...
There are a number of riboswitches that utilize the same ligand-binding domain to regulate either transcription or translation. S-box (SAM-I) riboswitches, including the riboswitch present in the Bacillus subtilis metI gene, which encodes cystathionine γ-synthase, regulate the expression of genes involved in methionine metabolism in response to SAM, primarily at the level of transcriptional attenuation. A rarer class of S-box riboswitches is predicted to regulate translation initiation. Here, we identified and characterized a translational S-box riboswitch in the metI gene from Desulfurispirillum indicum The regulatory mechanisms of riboswitches are influenced by the kinetics of ligand interaction. The half-life of the translational D. indicum metI RNA-SAM complex is significantly shorter than that of the transcriptional B. subtilis metI RNA. This finding suggests that unlike the transcriptional RNA, the translational metI riboswitch can make multiple reversible regulatory decisions. Comparison of both RNAs revealed that the second internal loop of helix P3 in the transcriptional RNA usually contains an A residue, whereas the translational RNA contains a C residue that is conserved in other S-box RNAs that are predicted to regulate translation. Mutational analysis indicated that the presence of an A or C residue correlates with RNA-SAM complex stability. These analyses indicate that the internal loop sequence critically determines the stability of the RNA-SAM complex by influencing the flexibility of residues involved in SAM binding and thereby affects the molecular mechanism of riboswitch function.
... In accordance with this lifestyle switch, the lactic acid bacteria are also characterized by an exceptional broad repertoire of carbon and amino acid transporters 56 , indicating that these bacteria often live in nutrient-rich environments. We postulate that the T-box regulation underlying some of these transporters might have evolved to accommodate the largely auxotrophic lifestyle of the lactic acid bacteria, where the T-box riboswitch might provide a more immediate regulatory response to amino acid starvation than the S-box riboswitch due to its specificity for methionine or any of the other auxotrophic amino acids 63,64 . ...
Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low- and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.
... The model for translation initiation, which is a rate-limiting step in translation, accurately predicted protein expression levels from mRNA sequences and enabled the design of mRNA sequences at desired expression levels. In metabolic engineering, control at the translational level has been applied to fine-tune the expression of proteins, instead of the expression of promoters (Vitreschak et al., 2004;Coppins et al., 2007;Kang et al., 2012;Na et al., 2013;Kang et al., 2014;Chae et al., 2015). For example, L-tyrosine production has been increased to 3.0 g/L by redesigning the 5 -UTR region in E. coli for optimizing the 5 -UTR of PEP synthetase (ppsA) . ...
Genome-scale engineering is a crucial methodology to rationally regulate microbiological system operations, leading to expected biological behaviors or enhanced bioproduct yields. Over the past decade, innovative genome modification technologies have been developed for effectively regulating and manipulating genes at the genome level. Here, we discuss the current genome-scale engineering technologies used for microbial engineering. Recently developed strategies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, multiplex automated genome engineering (MAGE), promoter engineering, CRISPR-based regulations, and synthetic small regulatory RNA (sRNA)-based knockdown, are considered as powerful tools for genome-scale engineering in microbiological systems. MAGE, which modifies specific nucleotides of the genome sequence, is utilized as a genome-editing tool. Contrastingly, synthetic sRNA, CRISPRi, and CRISPRa are mainly used to regulate gene expression without modifying the genome sequence. This review introduces the recent genome-scale editing and regulating technologies and their applications in metabolic engineering.
... This is accomplished by coordination between an aptamer domain, that binds the ligand, and an expression platform that translates this binding into downstream gene expression. Several riboswitches are identified across many phyla of bacteria [6,[35][36][37][38][39], and may be ancient in origin [40,41]. However, riboswitches have also evolved under varied environmental and genetic contexts. ...
In comparison to protein coding sequences, the impact of mutation and natural selection on the sequence and function of non-coding (ncRNA) genes is not well understood. Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be rapidly evolving. Compared to protein coding sequences, there are many challenges associated with assessment of ncRNAs that are not well addressed by conventional phylogenetic approaches, including: short sequence length, lack of primary sequence conservation, and the importance of secondary structure for biological function. Riboswitches are structured ncRNAs that directly interact with small molecules to regulate gene expression in bacteria. They typically consist of a ligand-binding domain (aptamer) whose folding changes drive changes in gene expression. The glycine riboswitch is among the most well-studied due to the widespread occurrence of a tandem aptamer arrangement (tandem), wherein two homologous aptamers interact with glycine and each other to regulate gene expression. However, a significant proportion of glycine riboswitches are comprised of single aptamers (singleton). Here we use graph clustering to circumvent the limitations of traditional phylogenetic analysis when studying the relationship between the tandem and singleton glycine aptamers. Graph clustering enables a broader range of pairwise comparison measures to be used to assess aptamer similarity. Using this approach, we show that one aptamer of the tandem glycine riboswitch pair is typically much more highly conserved, and that which aptamer is conserved depends on the regulated gene. Furthermore, our analysis also reveals that singleton aptamers are more similar to either the first or second tandem aptamer, again based on the regulated gene. Taken together, our findings suggest that tandem glycine riboswitches degrade into functional singletons, with the regulated gene(s) dictating which glycine-binding aptamer is conserved.
... Over 20 types of riboswitches have been identified by bioinformatics methods through sequence similarities. The ligands include metabolites such as purines, cofactors and their derivatives, amino acids, cations, anions, and antibiotics (Grundy and Henkin 2004;Vitreschak et al. 2004;Wickiser et al. 2005;Jia et al. 2013;Serganov and Nudler 2013). There are also classes of temperature sensing regulatory RNAs (Kortmann et al. 2011;Kortmann and Narberhaus 2012;Krajewski et al. 2014). ...
The 5' untranslated region (5' UTR) of eukaryotic mRNA plays important role in translation. Here we report the function of the 5' UTR mRNA of S-adenosylmethionine synthetase (sam1) in translational modulation in the presence of SAM in fission yeast Schizosaccharomyces pombe. Reporter assays, binding and chemical probing experiments and mutational analysis show that the 5' UTR mRNA of sam1 binds to SAM to effect translation. Translational modulation is dependent on a tertiary structure transition in the RNA upon SAM binding. The characterization of such an RNA that is directly associated with an essential metabolic process in eukaryotes provides additional evidence that ligand binding by RNAs play important roles in eukaryotic gene regulation.
Cyanobacteria are valuable resource for various bioactive compounds like pigments, sugars, lipids, amino acids, vitamins which have multifarious commercial applications. Further, their mass cultivation is less input intensive due to autotrophic nature. Despite such advantages, their commercial exploitation is not up to the mark mainly because of low inherent genetic potential for production of the target molecules. In depth understanding of the cyanobacterial metabolism at gene and metabolite level is required for redesigning the metabolic pathways and networks for improved production of target compounds. Overexpression, heterologous expression of effective genes, knocking out or silencing the genes coding for enzymes those divert metabolic fluxes toward undesirable products, improving transcriptional or translational efficiency, expressing designer genes are some popular approaches for metabolic engineering cyanobacterial strains. With the advent of “Omics” generated biological information and advance computing tools, it is now possible to develop metabolic models which can help to identify possible metabolic engineering targets at pathway and gene level. However, execution of such strategies requires genetic tool boxes which are yet to be developed for many of the commercially important strains. Nevertheless, there is no doubt that cyanobacterial-based bioeconomy of the future will definitely be driven by the metabolically redesigned strains.
Antimicrobial drug resistance has emerged as a significant challenge in contemporary medicine due to the proliferation of numerous bacterial strains resistant to all existing antibiotics. Meanwhile, riboswitches have emerged as promising targets for discovering antibacterial drugs. Ri-boswitches are regulatory elements in certain bacterial mRNAs that can bind to specific molecules and control gene expression via transcriptional termination, prevention of translation, or mRNA destabilization. By targeting riboswitches, we aim to develop innovative strategies to combat antibiotic-resistant bacteria and enhance the efficacy of antibacterial treatments. This convergence of challenges and opportunities underscores the ongoing quest to revolutionize medical approaches against evolving bacterial threats. For the first time, this innovative review describes the rational design and applications of chimeric antisense oligonucleotides as antibacterial agents targeting four riboswitches selected based on genome-wide bioinformatic analyses. The antisense oligonucleotides are coupled with the cell-penetrating oligopeptide pVEC, which penetrates Gram-positive and Gram-negative bacteria and specifically targets glmS, FMN, TPP, and SAM-I riboswitches in Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli. The average antibiotic dosage of antisense oligonucleotides that inhibits 80% of bacterial growth is around 700 nM (4.5 µg/mL). Antisense oligonucleotides do not exhibit toxicity in human cell lines at this concentration. The results demonstrate that these riboswitches are suitable targets for antibacterial drug development using antisense oligonucleotide technology. The approach is fully rational because selecting suitable riboswitch targets and designing ASOs that target them are based on predefined criteria. The approach can be used to develop narrow or broad-spectrum antibiotics against multidrug-resistant bacterial strains for a short time. The approach is easily adaptive to new resistance using targeting NGS technology.
Riboswitches have received significant attention over the last two decades for their multiple functionalities and great potential for applications in various fields. This article highlights and reviews the recent advances in biosensing and biotherapy. These fields involve a wide range of applications, such as food safety detection, environmental monitoring, metabolic engineering, live cell imaging, wearable biosensors, antibacterial drug targets, and gene therapy. The discovery, origin, and optimization of riboswitches are summarized to help readers better understand their multidimensional applications. Finally, this review discusses the multidimensional challenges and development of riboswitches in order to further expand their potential for novel applications.
Riboswitches are conserved structural ribonucleic acid (RNA) sensors that are mainly found to regulate a large number of genes/operons in bacteria. Presently, >50 bacterial riboswitch classes have been discovered, but only the thiamine pyrophosphate riboswitch class is detected in a few eukaryotes like fungi, plants and algae. One of the most important challenges in riboswitch research is to discover existing riboswitch classes in eukaryotes and to understand the evolution of bacterial riboswitches. However, traditional search methods for riboswitch detection have failed to detect eukaryotic riboswitches besides just one class and any distant structural homologs of riboswitches. We developed a novel approach based on inverse RNA folding that attempts to find sequences that match the shape of the target structure with minimal sequence conservation based on key nucleotides that interact directly with the ligand. Then, to support our matched candidates, we expanded the results into a covariance model representing similar sequences preserving the structure. Our method transforms a structure-based search into a sequence-based search that considers the conservation of secondary structure shape and ligand-binding residues. This method enables us to identify a potential structural candidate in fungi that could be the distant homolog of bacterial purine riboswitches. Further, phylogenomic analysis and evolutionary distribution of this structural candidate indicate that the most likely point of origin of this structural candidate in these organisms is associated with the loss of traditional purine riboswitches. The computational approach could be applicable to other domains and problems in RNA research.
Deep learning is a class of machine learning techniques capable of creating internal representation of data without explicit preprogramming. Hence, in addition to practical applications, it is of interest to analyze what features of biological data may be learned by such models. Here, we describe PredPair, a deep learning neural network trained to predict base pairs in RNA structure from sequence alone, without any incorporated prior knowledge, such as the stacking energies or possible spatial structures. PredPair learned the Watson-Crick and wobble base-pairing rules and created an internal representation of the stacking energies and helices. Application to independent experimental (DMS-Seq) data on nucleotide accessibility in mRNA showed that the nucleotides predicted as paired indeed tend to be involved in the RNA structure. The performance of the constructed model was comparable with the state-of-the-art method based on the thermodynamic approach, but with a higher false positives rate. On the other hand, it successfully predicted pseudoknots. t-SNE clusters of embeddings of RNA sequences created by PredPair tend to contain embeddings from particular Rfam families, supporting the predictions of PredPair being in line with biological classification.
Riboflavin is an essential nutrient for humans and animals, and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are cofactors in the cells. Therefore, riboflavin and its derivatives are widely used in the food, pharmaceutical, nutraceutical and cosmetic industries. Advances in biotechnology have led to a complete shift in the commercial production of riboflavin from chemical synthesis to microbial fermentation. In this review, we provide a comprehensive review of biotechnologies that enhance riboflavin production in microorganisms, as well as representative examples. Firstly, the synthesis pathways and metabolic regulatory processes of riboflavin in microorganisms; and the current strategies and methods of metabolic engineering for riboflavin production are systematically summarized and compared. Secondly, the using of systematic metabolic engineering strategies to enhance riboflavin production is discussed, including laboratory evolution, histological analysis and high-throughput screening. Finally, the challenges for efficient microbial production of riboflavin and the strategies to overcome these challenges are prospected.
Modeling the three-dimensional structure of RNAs is a milestone toward better understanding and prediction of nucleic acids molecular functions. Physics-based approaches and molecular dynamics simulations are not tractable on large molecules with all-atom models. To address this issue, coarse-grained models of RNA three-dimensional structures have been developed. In this chapter, we describe a graphical modeling based on the Leontis–Westhof extended base pair classification. This representation of RNA structures enables us to identify highly conserved structural motifs with complex nucleotide interactions in structure databases. We show how to take advantage of this knowledge to quickly predict three-dimensional structures of large RNA molecules and present the RNA-MoIP web server (http://rnamoip.cs.mcgill.ca) that streamlines the computational and visualization processes. Finally, we show recent advances in the prediction of local 3D motifs from sequence data with the BayesPairing software and discuss its impact toward complete 3D structure prediction.
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential cofactors for enzymes, which catalyze a broad spectrum of vital reactions. This paper intends to compile all potential FAD/FMN-binding proteins encoded by the genome of Arabidopsis thaliana. Several computational approaches were applied to group the entire flavoproteome according to (i) different catalytic reactions in enzyme classes, (ii) the localization in subcellular compartments, (iii) different protein families and subclasses, and (iv) their classification to structural properties. Subsequently, the physiological significance of several of the larger flavoprotein families was highlighted. It is conclusive that plants, such as Arabidopsis thaliana, use many flavoenzymes for plant-specific and pivotal metabolic activities during development and for signal transduction pathways in response to biotic and abiotic stress. Thereby, often two up to several homologous genes are found encoding proteins with high protein similarity. It is proposed that these gene families for flavoproteins reflect presumably their need for differential transcriptional control or the expression of similar proteins with modified flavin-binding properties or catalytic activities.
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential cofactors for enzymes, which catalyze a broad spectrum of vital reactions. This paper intends to compile all potential FAD/FMN-binding proteins encoded by the genome of Arabidopsis thaliana. Several computational approaches were applied to group the entire flavoproteome according to (i) different catalytic reactions in enzyme classes, (ii) the localization in subcellular compartments, (iii) different protein families and subclasses, and (iv) their classification to structural properties. Subsequently, the physiological significance of several of the larger flavoprotein families was highlighted. It is conclusive that plants, such as Arabidopsis thaliana, use many flavoenzymes for plant-specific and pivotal metabolic activities during development and for signal transduction pathways in response to biotic and abiotic stress. Thereby, often two up to several homologous genes are found encoding proteins with high protein similarity. It is proposed that these gene families for flavoproteins reflect presumably their need for differential transcriptional control or the expression of similar proteins with modified flavin-binding properties or catalytic activities.
Fundamentally, riboswitches are known to be highly structured and conserved metabolite binding domains, known to be aptamer, which are locus within mRNAs. When we talk about RNA world, riboswitches are ranked among the trending topics. Since its discovery from 2002, there is a large amount of feature disclosed till now and thanks to computational analysis this finding are increasing every year with a fast rate. This work club up the information regarding new findings within the field of riboswitches, which include data about its new classes discovery and its application in the field of gene regulation mechanism, medical science, inter-cellular signalling, biosensors and many more.
Synthetic biology is a newly growing field which allows us to design non-natural parts, devices and circuits for biotechnological applications. These novel systems can help to find a solution for current challenges that we are facing in context to fulfilling the demand of drugs, vaccines, precise diagnosis, fine chemicals, biofuels and so on. In the past decade, a number of parts, devices and systems have been engineered and characterized in many organisms. Currently, a number of research groups are focusing on the development of new technologies/assays, including CRISPR-Cas9, riboregulators, riboswitches, cell-free protein synthesis and microfluidics that can accelerate synthetic biology research and its applications. This chapter highlights the progress, challenges and applications of parts, devices, circuits and tools towards biological, biomedical, therapeutic and industrial purpose.
The theophylline aptamer was isolated from an oligonucleotide library in 1994. Since that time, the aptamer has found wide utility, particularly in synthetic biology, metabolic engineering, and diagnostic applications. The primary application of the theophylline aptamer is in the construction and characterization of synthetic riboswitches for regulation of gene expression. These riboswitches have been used to control cellular motility, regulate carbon metabolism, construct logic gates, screen for mutant enzymes, and control apoptosis. Other applications of the theophylline aptamer in cellular engineering include regulation of RNA interference and genome editing through CRISPR systems. Here we describe the uses of the theophylline aptamer for cellular engineering over the past 25 years. In so doing, we also highlight important synthetic biology applications to control gene expression in a ligand-dependent manner.
Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs primarily by an attenuation control mechanism. Previous studies have suggested a model for attenuation control in which low intracellular levels of UTP cause close coupling of transcription and translation within the pyrBI leader region. This close coupling apparently prevents transcriptional termination at an attenuator (a rho-independent transcriptional terminator) located 23 base pairs before the pyrBI structural genes within an open reading frame for a 44-amino acid leader polypeptide. Presumably, a ribosome involved in the synthesis of the leader polypeptide disrupts or precludes the formation of the attenuator-encoded RNA hairpin, which is required for transcriptional termination. In this study, we examined the role of the ribosome in inhibiting transcriptional termination at the pyrBI attenuator. Using oligonucleotide-directed mutagenesis, we systematically introduced termination codons into the reading frame for the leader polypeptide to determine the distance a ribosome must translate to suppress transcriptional termination. These mutations were incorporated individually into a pyrB::lacZ gene fusion, which was then introduced into the E. coli chromosome. The resulting fusion strains were used to measure the effect of each mutation on pyrB::lacZ expression. The results show that a ribosome must translate to within 14-16 nucleotides of the attenuator-encoded RNA hairpin to inhibit transcriptional termination efficiently, which indicates a direct interaction between the ribosome and the termination hairpin sequence as proposed in the present model. Additional results indicate that factors not included in the present model for attenuation control contribute to the expression and regulation of the pyrBI operon.
This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.
A 3.5 kb EcoRI-BamHI fragment of Bacillus subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-Escherichia coli shuttle vector pCB20. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of micro-organisms, such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of flavokinase activity in cell extracts demonstrated that ribR encodes a monofunctional flavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.
Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.
Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and
expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences
of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity.
Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate
ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand
association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity.
These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to
many cellular processes involving nucleic acids.
The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5' regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helices, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.
tion affects the activity of the promoter and the amount of mRNA made. Regu- lation by thiamin is shown to be posttran- scription initiation (1). The amount of mRNA initiated from the promoter ap- pears unaffected by the presence of thia- min, but the elongation of the mRNA is attenuated by a terminator structure in the middle of the first gene of the operon, thiC .I nBacillus subtilis, the trp operon is regulated by a similar attenuation process (2, 3). When the TRAP protein binds tryptophan, it can then bind to the mRNA and lead to its premature termination, inhibiting the expression of all the genes required for tryptophan synthesis. E. coli also uses an attenuation process as a sec- ond level of control, with the ribosome sensing the concentration of tryptophan in the cell (actually the concentration of charged tRNATrp) (2). In thiamin regulation, the attenuating termination structure forms when trans- lation initiation of the thiC gene is blocked (1). When the mRNA is trans- lated by a ribosome, the structure will not form, but thiamin somehow inhibits ribosome initia- tion, thereby lead-
The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subject to control by the purR gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the purA and purR genes. A consensus sequence for the binding of PurR, named the PurBox, has been suggested (M. Kilstrup, S. G. Jessing, S.
B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900–3906, 1998). To determine whether the expression
of other genes might be regulated by PurR, we performed a search for PurBox sequences in the B. subtilis genome sequence and found several candidate PurBoxes. By the use of transcriptionallacZ fusions, five selected genes or operons (glyA, yumD, yebB,xpt-pbuX, and yqhZ-folD), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR. Using a machine-learning
algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two
PurBoxes in their upstream control regions. The two boxes are divergently oriented, forming a palindromic sequence with the
inverted repeats separated by 16 or 17 nucleotides. A computerized search revealed one additional PurR-regulated gene,ytiP. The significance of the tandem PurBox motifs was demonstrated in vivo by deletion analysis and site-directed mutagenesis
of the two PurBox sequences located upstream of glyA. All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism. Functional analysis
showed thatyebB encodes the previously characterized hypoxanthine-guanine permease PbuG and that ytiP encodes another guanine-hypoxanthine permease and is now named pbuO. yumD encodes a GMP reductase and is now namedguaC.
We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology. Quality control tests of the B. subtilis GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization
conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression
patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known
open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated
by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite
coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.
There are two major pathways for methionine biosynthesis in micro-organisms. Little is known about these pathways in Bacillus subtilis. The authors assigned a function to the metI (formerly yjcI) and metC (formerly yjcJ) genes of B. subtilis by complementing Escherichia coli metB and metC mutants, analysing the phenotype of B. subtilis metI and metC mutants, and carrying out enzyme activity assays. These genes encode polypeptides belonging to the cystathionine gamma-synthase family of proteins. Interestingly, the MetI protein has both cystathionine gamma-synthase and O-acetylhomoserine thiolyase activities, whereas the MetC protein is a cystathionine beta-lyase. In B. subtilis, the transsulfuration and the thiolation pathways are functional in vivo. Due to its dual activity, the MetI protein participates in both pathways. The metI and metC genes form an operon, the expression of which is subject to sulfur-dependent regulation. When the sulfur source is sulfate or cysteine the transcription of this operon is high. Conversely, when the sulfur source is methionine its transcription is low. An S-box sequence, which is located upstream of the metI gene, is involved in the regulation of the metIC operon. Northern blot experiments demonstrated the existence of two transcripts: a small transcript corresponding to the premature transcription termination at the terminator present in the S-box and a large one corresponding to transcription of the complete metIC operon. When methionine levels were limiting, the amount of the full-length transcript increased. These results substantiate a model of regulation by transcription antitermination.
The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements.
A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested. In Gram‐positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most
Gram‐negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation. Several
new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae). Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with
the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram‐positive bacterium; non‐regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene.
Biotin is a necessary cofactor of numerous biotin-dependent carboxylases in a variety of microorganisms. The strict control of biotin biosynthesis in Escherichia coli is mediated by the bifunctional BirA protein, which acts both as a biotin-protein ligase and as a transcriptional repressor of the biotin operon. Little is known about regulation of biotin biosynthesis in other bacteria. Using comparative genomics and phylogenetic analysis, we describe the biotin biosynthetic pathway and the BirA regulon in most available bacterial genomes. Existence of an N-terminal DNA-binding domain in BirA strictly correlates with the presence of putative BirA-binding sites upstream of biotin operons. The predicted BirA-binding sites are well conserved among various eubacterial and archaeal genomes. The possible role of the hypothetical genes bioY and yhfS-yhfT, newly identified members of the BirA regulon, in the biotin metabolism is discussed. Based on analysis of co-occurrence of the biotin biosynthetic genes and bioY in complete genomes, we predict involvement of the transmembrane protein BioY in biotin transport. Various nonorthologous substitutes of the bioC-coupled gene bioH from E. coli, observed in several genomes, possibly represent the existence of different pathways for pimeloyl-CoA biosynthesis. Another interesting result of analysis of operon structures and BirA sites is that some biotin-dependent carboxylases from Rhodobacter capsulatus, actinomycetes, and archaea are possibly coregulated with BirA. BirA is the first example of a transcriptional regulator with a conserved binding signal in eubacteria and archaea.
Vitamin B1 in its active form thiamin pyrophosphate is an essential coenzyme that is synthesized by coupling of pyrimidine (hydroxymethylpyrimidine;
HMP) and thiazole (hydroxyethylthiazole) moieties in bacteria. Using comparative analysis of genes, operons, and regulatory
elements, we describe the thiamin biosynthetic pathway in available bacterial genomes. The previously detected thiamin-regulatory
element,thi box (Miranda-Rios, J., Navarro, M., and Soberon, M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9736–9741), was extended, resulting in a new, highly conserved RNA secondary structure, the THI element, which is widely distributed in eubacteria and also occurs in some archaea. Search for THIelements and analysis of operon structures identified a large number of new candidate thiamin-regulated genes, mostly transporters,
in various prokaryotic organisms. In particular, we assign the thiamin transporter function to yuaJ in theBacillus/Clostridium group and the HMP transporter function to an ABC transporter thiXYZ in some proteobacteria and firmicutes. By analogy to the model of regulation of the riboflavin biosynthesis, we suggest thiamin-mediated
regulation based on formation of alternative RNA structures involving theTHI element. Either transcriptional or translational attenuation mechanism may operate in different taxonomic groups, dependent
on the existence of putative hairpins that either act as transcriptional terminators or sequester translation initiation sites.
Based on analysis of co-occurrence of the thiamin biosynthetic genes in complete genomes, we predict that eubacteria, archaea,
and eukaryota have different pathways for the HMP and hydroxyethylthiazole biosynthesis.
Vitamins B12, B6, and folic acid converge at the homocysteine metabolic junction where they support the activities of two key enzymes involved in intracellular homocysteine management, methionine synthase (MS) and cystathionine beta-synthase. The molecular mechanism for the regulation of homocysteine metabolism by B12 supplementation has been investigated in this study. B12 supplementation does not alter mRNA or protein turnover rates but induces translational up-regulation of MS by shifting the mRNA from the ribonucleoprotein to the polysome pool. The B12-responsive element has been localized by deletion analysis using a reporter gene assay to a 70-bp region located at the 3' end of the 5'-untranslated region of the MS mRNA. The cellular consequence of the B12 response is a 2- and 3.5-fold increase in the flux of homocysteine through the MS-dependent transmethylation pathway in HepG2 and 293 cells, respectively. It is speculated that B12-induced up-regulation of MS may have evolved as an adaptive strategy for rapidly sequestering an essential and rare nutrient whose availability may have been limited in the evolutionary history of mammals, a problem that is exacerbated by the absence of this vitamin from the plant kingdom.
Many operons in Gram-positive bacteria that are involved in methionine (Met) and cysteine (Cys) biosynthesis possess an evolutionarily conserved regulatory leader sequence (S-box) that positively controls these genes in response to methionine starvation. Here, we demonstrate that a feed-back regulation mechanism utilizes S-adenosyl-methionine as an effector. S-adenosyl-methionine directly and specifically binds to the nascent S-box RNA, causing an intrinsic terminator to form and interrupt transcription prematurely. The S-box leader RNA thus expands the family of newly discovered riboswitches, i.e., natural regulatory RNA aptamers that seem to sense small molecules ranging from amino acid derivatives to vitamins.
In Bacillus subtilis expression of genes or operons encoding enzymes and other proteins involved in purine synthesis is affected by purine bases
and nucleosides in the growth medium. The genes belonging to the PurR regulon (purR, purA, glyA, guaC, pbuO, pbuG, and the pur, yqhZ-folD, and xpt-pbuX operons) are controlled by the PurR repressor, which inhibits transcription initiation. Other genes are regulated by a less-well-described
transcription termination mechanism that responds to the presence of hypoxanthine and guanine. The pur operon and the xpt-pbuX operon, which were studied here, are regulated by both mechanisms. We isolated two mutants resistant to 2-fluoroadenine in
which the pur operon and the xpt-pbuX operon are expressed at increased levels in a PurR-independent manner. The mutations were caused by deletions that disrupted
a potential transcription terminator structure located immediately upstream of the ydhL gene. The 5′ part of the ydhL leader region contained a 63-nucleotide (nt) sequence very similar to the 5′ ends of the leaders of the pur and xpt-pbuX operons. Transcripts of these regions may form a common tandem stem-loop secondary structure. Two additional genes with potential
leader regions containing the 63-nt sequence are pbuG, encoding a hypoxanthine-guanine transporter, and yxjA, which was shown to encode a purine nucleoside transporter and is renamed nupG. Transcriptional lacZ fusions and mutations in the 63-nt sequence encoding the possible secondary structures provided evidence that expression
of the pur and xpt-pbuX operons and expression of the ydhL, nupG, and pbuG genes are regulated by a common mechanism. The new pur regulon is designated the XptR regulon. Except for ydhL, the operons and genes were negatively regulated by hypoxanthine and guanine. ydhL was positively regulated. The derived amino acid sequence encoded by ydhL (now called pbuE) is similar to the amino acid sequences of metabolite efflux pumps. When overexpressed, PbuE lowers the sensitivity to purine
analogs. Indirect evidence indicated that PbuE decreases the size of the internal pool of hypoxanthine. This explains why
the hypoxanthine- and guanine-regulated genes are expressed at elevated levels in a mutant that overexpresses pbuE.
Comparative analysis of genes, operons and regulatory elements was applied to the lysine biosynthetic pathway in available
bacterial genomes. We report identification of a lysine‐specific RNA element, named the LYS element, in the regulatory regions of bacterial genes involved in biosynthesis and transport of lysine. Similarly to the
previously described RNA regulatory elements for three vitamins (riboflavin, thiamin and cobalamin), purine and methionine
regulons, this regulatory RNA structure is highly conserved on the sequence and structural levels. The LYS element includes regions of lysine‐constitutive mutations previously identified in Escherichia coli and Bacillus subtilis. A possible mechanism of the lysine‐specific riboswitch is similar to the previously defined mechanisms for the other metabolite‐specific
riboswitches and involves either transcriptional or translational attenuation in various groups of bacteria. Identification
of LYS elements in Gram‐negative γ‐proteobacteria, Gram‐positive bacteria from the Bacillus/Clostridium group, and Thermotogales resulted in description of the previously uncharacterized lysine regulon in these bacterial species.
Positional analysis of LYS elements led to identification of a number of new candidate lysine transporters, namely LysW, YvsH and LysXY. Finally, the
most likely candidates for genes of lysine biosynthesis missing in Gram‐ positive bacteria were identified using the genome
context analysis.
Cobalamin in the form of adenosylcobalamin (Ado-CBL) is known to repress expression of genes for vitamin B12 biosynthesis and be transported by a posttranscriptional regulatory mechanism, which involves direct binding of Ado-CBL to 5′untranslated gene regions (5′UTR). Using comparative analysis of genes and regulatory regions, we identified a highly conserved RNA structure, the B12-element, which is widely distributed in 5′UTRs of vitamin B12-related genes in eubacteria. Multiple alignment of approximately 200 B12-elements from 66 bacterial genomes reveals their common secondary structure and several extended regions of sequence conservation, including the previously known B12-box motif. In analogy to the model of regulation of the riboflavin and thiamin biosynthesis, we suggest Ado-CBL-mediated regulation based on formation of alternative RNA structures including the B12-element. In Gram-negative proteobacteria, as well as in cyanobacteria, actinobacteria, and the CFB group, the cobalamin biosynthesis and vitamin B12 transport genes are predicted to be regulated by inhibition of translation initiation, whereas in the Bacillus/Clostridium group of Gram-positive bacteria, these genes seem to be regulated by transcriptional antitermination. Phylogenetic analysis of the B12-elements reveals a large number of likely duplications of B12-elements in several bacterial genomes. These lineage-specific duplications of RNA regulatory elements seem to be a major evolutionary mechanism for expansion of the vitamin B12 regulon.
Keywords
• Bacteria
• comparative genomics
• regulatory RNA
• B12-element
• cobalamin
RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 1012 random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable
secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA)
of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 µM by equilibrium
filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil.
The Kd values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand
are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the
addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the
consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin
ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding.
Induction of the Bacillus subtilis sacB gene and sacPA operon and Escherichia coli bgl operon is mediated by structurally homologous antiterminators encoded by the sacY, sacT, and bglG genes, respectively. When activated, these proteins prevent early transcription termination at terminators located in the leader regions of the three operons. BglG was previously shown to bind in vitro to an imperfectly palindromic 29-nucleotide RNA sequence located upstream of the terminator and partially overlapping with it [Houman, F., Diaz-Torres, M.R. & Wright, A. (1990) Cell 62, 1153-1163]. Similar motifs, here termed ribonucleic antiterminators (RATs), strongly conserved in sequence and in position, are found in the leader of both sacB and sacPA. Mutations were created in sacB RAT and tested in B. subtilis; this showed that sacB RAT is the target for SacY-mediated induction of sacB and that a stem-loop structure in the mRNA is required for regulatory function. Mutations increasing the similarity of the sacB RAT with those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively; most of these changes did not strongly affect induction by SacY, suggesting that the nucleotides at these variable positions act as negative specificity determinants.
This chapter discusses the recent progress made in understanding de novo purine nucleotide biosynthesis and its regulation in bacteria. A purR-encoded repressor—operator interaction plays a key role in the genetic regulation of purine biosynthesis in E. coli and also appears to be involved in coordinating the expression of genes in related metabolic pathways. The outline of a more complex regulatory system has been described for de novo purine nucleotide synthesis in B. subtilis. These advances have resulted from the cloning and analyses of structural genes encoding the biosynthetic enzymes and the purR regulatory gene from E. coli. The cloning of regulatory genes is essential for a further understanding of the regulation of purine nucleotide biosynthesis in B. subtilis.
We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.
The expression of the Bacillus subtilis lysC operon, which encodes the first specific enzyme of lysine biosynthesis, is controlled by the availability of the end product, lysine. The question of whether lysine exerts its control by inducing premature termination of transcription was addressed using Northern blot analysis. Whereas lys-C-specific RNA from lysine-starved B. subtilis consisted primarily of the expected full-length mRNA (1.6 kb), that from bacteria grown with an excess of lysine consisted of a truncated 0.27 kb RNA in place of the full-length 1.6 kb transcript. On the other hand, a B. subtilis aecA mutant, in which the lysC operon was derepressed owing to a single nucleotide substitution in the region corresponding to the lysC leader transcript, produced full-length lysC mRNA, but no 0.27 kb RNA, even during growth with excess lysine. Mapping of the truncated 0.27 kb lysC RNA by hybridization with oligonucleotide probes showed that it corresponded to the upstream portion of the lysC leader transcript, extending from the transcription initiation site to a putative rho-independent terminator element. Quantitative transcript analysis by hybridization with specific oligonucleotides showed that lysine did not affect the number of lysC-specific RNA molecules but promoted the stoichiometric replacement of full-length mRNA with truncated 0.27 kb molecules. These results indicate that lysine regulates the expression of the lysC operon by effecting the premature termination of transcription at a rho-independent terminator site in the lysC leader region and that the site of the aecA mutation, far upstream of the putative terminator element, must play an essential role in premature transcription termination by a mechanism which is not yet understood.
The xpt and pbuX genes from Bacillus subtilis were cloned, and their nucleotide sequences were determined. The xpt gene encodes a specific xanthine phosphoribosyltransferase, and the pbuX gene encodes a xanthine-specific purine permease. The genes have overlapping coding regions, and Northern (RNA) blot analysis indicated an operon organization. The translation of the second gene, pbuX, was strongly dependent on the translation of the first gene, xpt. Expression of the operon was repressed by purines, and the effector molecules appear to be hypoxanthine and guanine. When hypoxanthine and guanine were added together, a 160-fold repression was observed. The regulation of expression was at the level of transcription, and we propose that a transcription termination-antitermination control mechanism similar to the one suggested for the regulation of the purine biosynthesis operon exists. The expression of the xpt-pbuX operon was reduced when hypoxanthine served as the sole nitrogen source. Under these conditions, the level of the hypoxanthine- and xanthine-degrading enzyme, xanthine dehydrogenase, was induced more than 80-fold. The xanthine dehydrogenase level was completely derepressed in a glnA (glutamine synthetase) genetic background. Although the regulation of the expression of the xpt-pbuX operon was found to be affected by the nitrogen source, it was normal in a glnA mutant strain. This result suggests the existence of different signalling pathways for repression of the transcription of the xpt-pbuX operon and the induction of xanthine dehydrogenase.
The Bacillus subtilis tryptophan biosynthetic genes are regulated by TRAP. Radiographic crystallography indicates that the TRAP complex contains 11 identical subunits arranged in a doughnut-like structure termed the beta-wheel. The trpEDCFBA operon is regulated by an attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the trp leader transcript. TRAP binding blocks formation of an anti-terminator structure, thereby promoting the formation of an overlapping terminator, resulting in transcription termination preceding the structural genes. When TRAP is not activated, it is unable to bind to the transcript, which allows anti-terminator formation and, hence, transcription of the operon. TRAP is also responsible for regulating translation of trpEand trpG. TRAP binding to trp operon readthrough transcripts promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence is sequestered in a hairpin, thus inhibiting TrpE synthesis. In the case of trpG, TRAP binds to nine repeats that overlap the ribosome-binding site, thereby blocking translation.
Short RNA aptamers that specifically bind to a wide variety of ligands in vitro can be isolated from randomized pools of RNA. Here it is shown that small molecule aptamers also bound their ligand in vivo, enabling development of a method for controlling gene expression in living cells. Insertion of a small molecule aptamer into the 5' untranslated region of a messenger RNA allowed its translation to be repressible by ligand addition in vitro as well as in mammalian cells. The ability of small molecules to control expression of specific genes could facilitate studies in many areas of biology and medicine.
The molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis. Analyses of the recently completed B. subtilis genome revealed 11 copies of a highly conserved motif. In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram-positive genera. The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription. The 5' portion of all of the leaders could fold into a conserved complex structure. Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator. Mutation of conserved primary sequence and structural elements supported a model in which the 5' portion of the leader forms an anti-antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti-antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available. This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine.
Nucleic acid receptors ('aptamers'), which recognize a large variety of organic molecules of low molecular weight, have been isolated from combinatorial nucleic acid libraries by in vitro selection methods. Structural studies of nucleic acid-small molecule complexes provide insight into both the principles of molecular recognition by this class of biopolymers and the architecture of tertiary motifs in nucleic acid folding. Aptamers that recognize small molecules are increasingly applied as tools in molecular biology, from the detection of oxidative damage in DNA to conditional gene expression and from their use as modules for the engineering of allosteric ribozymes to biosensors.
This work was partially supported by grants from the Russian State ‘Human Genome’ program and the Russian Fund of Basic Research under grant 99-04-48347. Preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org.
Availability of complete bacterial genomes opens the way to the comparative approach to the recognition of transcription regulatory sites. Assumption of regulon conservation in conjunction with profile analysis provides two lines of independent evidence making it possible to make highly specific predictions. Recently this approach was used to analyze several regulons in eubacteria and archaebacteria. The present review covers recent advances in the comparative analysis of transcriptional regulation in prokaryotes and phylogenetic fingerprinting techniques in eukaryotes, and describes the emerging patterns of the evolution of regulatory systems.
Expression of the btuB gene encoding the outer membrane cobalamin transporter in Escherichia coli is strongly reduced on growth with cobalamins. Previous studies have shown that this regulation occurs in response to adenosylcobalamin (Ado-Cbl) and operates primarily at the translational level. Changes in the level and stability of btuB RNA are consequences of the modulated translation initiation. To examine how Ado-Cbl affects translation, the binding of E. coli 30S ribosomal subunits to btuB RNA was investigated by using a primer extension inhibition assay. Ribosome binding to btuB RNA was much less efficient than to other RNAs and was preferentially lost when the ribosomes were subjected to a high-salt wash. Ribosome binding to btuB RNA was inhibited by Ado-Cbl but not by cyanocobalamin, with half-maximal inhibition around 0.3 microM Ado-Cbl. Ribosome-binding activity was increased or decreased by mutations in the btuB leader region, which affected two predicted RNA hairpins and altered expression of btuB-lacZ reporters. Finally, the presence of Ado-Cbl elicited formation of a single primer extension-inhibition product with the same specificity and Cbl-concentration dependence as the inhibition of ribosome binding. These results indicate that btuB expression is controlled by the specific binding of Ado-Cbl to btuB RNA, which then affects access to its ribosome-binding sequence.
Expression of the cobalamin (Cbl) biosynthetic cob operon in Salmonella typhimurium is repressed by the end-product. This regulation is conferred mainly at the translational level and involves a cobalamin-induced folding of an RNA hairpin that sequesters the ribosomal binding site (RBS) of the cob mRNA and prevents translation initiation. A combined structural and mutational analysis shows that a cis-acting translational enhancer (TE) element, located 83 nucleotides upstream of the Shine-Dalgarno sequence in the 5'-untranslated region (5'-UTR) of the cob mRNA, is required to unfold the inhibitory RBS hairpin in the absence of cobalamin. The TE element, which consists of 5 nucleotides, is proposed to confer its enhancer function in the absence of cobalamin by interacting with nucleotides in the stem of the RBS hairpin. This interaction destabilizes the RNA hairpin and allows ribosome binding. In the presence of cobalamin, the enhancer function is inhibited. As a result, the RBS hairpin forms and prevents translation initiation. Several additional RNA hairpins in the 5'-UTR were also identified and are suggested to be important for repression. The above data suggest that normal cobalamin repression of the cob operon requires that the 5'-UTR has a defined secondary and tertiary structure.
Previous studies have shown that the introduction of a ligand-binding RNA (aptamer) into the 5'-UTR of an mRNA can confer regulated expression of both prokaryotic and eukaryotic reporter genes. The current report shows that aptamer insertion into the 5'-UTR of a cyclin transcript in S. cerevisiae renders cell-cycle control dependent upon the presence or absence of the target ligand. A malachite green binding motif, defined by an asymmetric internal loop flanked by short RNA helices, was inserted immediately upstream of the CLB2 start codon. Progression through the cell cycle is dramatically slowed and elongated bud morphology develops when tetramethylrosamine (a fluorescent malachite green analogue) is added to the aptamer-containing strain. Quantification of CLB2 expression at the RNA and protein levels by RT-PCR and Western blot analysis, respectively, demonstrates that the aptamer ligand regulates transcript translatability rather than stability. One-dimensional NMR spectroscopy shows that the malachite green binding aptamer undergoes a dramatic ligand-dependent change in structure with many nucleotides folding to adopt a well-defined conformation. These results are consistent with a model in which translational initiation is blocked by ligand-induced conformational changes in the 5'-UTR.
The T box transcription termination control system is used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA
synthetase and other amino acid-related genes. Readthrough of a transcriptional terminator located in the leader region of
the target gene is dependent on a specific interaction between the nascent leader transcript and the cognate uncharged tRNA.
This interaction is required for formation of an antiterminator structure in the leader, which prevents formation of a competing
transcriptional terminator stem–loop. The antiterminators and terminators of genes in this family are highly conserved in
both secondary structure and primary sequence; the antiterminator contains the T box sequence, which is the most highly conserved
leader element. These conserved features were investigated by phylogenetic and mutational analysis. Changes at highly conserved
positions in the bulge and in the helix above the bulge reduced function, while alteration of other positions that were as
much as 96% conserved did not have a major effect. The disparity between sequence conservation and function may be due to
the requirement for maintaining base pairing in both the antiterminator and terminator structures.
Regulation of gene expression by premature termination of transcription, or transcription attenuation, is a common regulatory strategy in bacteria. Various mechanisms of regulating transcription termination have been uncovered, each can be placed in either of two broad categories of termination events. Many mechanisms involve choosing between two alternative hairpin structures in an RNA transcript, with the decision dependent on interactions between ribosome and transcript, tRNA and transcript, or protein and transcript. In other examples, modification of the transcription elongation complex is the crucial event. This article will describe and compare several of these regulatory strategies, and will cite specific examples to illustrate the different mechanisms employed.
Messenger RNAs are typically thought of as passive carriers of genetic information that are acted upon by protein- or small RNA-regulatory factors and by ribosomes during the process of translation. We report that the 5'-untranslated sequence of the Escherichia coli btuB mRNA assumes a more proactive role in metabolic monitoring and genetic control. The mRNA serves as a metabolite-sensing genetic switch by selectively binding coenzyme B(12) without the need for proteins. This binding event establishes a distinct RNA structure that is likely to be responsible for inhibition of ribosome binding and consequent reduction in synthesis of the cobalamin transport protein BtuB. This finding, along with related observations, supports the hypothesis that metabolic monitoring through RNA-metabolite interactions is a widespread mechanism of genetic control.
Although proteins fulfil most of the requirements that biology has for structural and functional components such as enzymes and receptors, RNA can also serve in these capacities. For example, RNA has sufficient structural plasticity to form ribozyme and receptor elements that exhibit considerable enzymatic power and binding specificity. Moreover, these activities can be combined to create allosteric ribozymes that are modulated by effector molecules. It has also been proposed that certain messenger RNAs might use allosteric mechanisms to mediate regulatory responses depending on specific metabolites. We report here that mRNAs encoding enzymes involved in thiamine (vitamin B(1)) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the need for protein cofactors. The mRNA-effector complex adopts a distinct structure that sequesters the ribosome-binding site and leads to a reduction in gene expression. This metabolite-sensing regulatory system provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.
The RFN element is a highly conserved domain that is found frequently in the 5'-untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport proteins. We report that this domain serves as the receptor for a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. Our results also indicate that in Bacillus subtilis, the riboswitch most likely controls gene expression by causing premature transcription termination of the ribDEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA. Sequence and structural analyses indicate that the RFN element is a natural FMN-binding aptamer, the allosteric character of which is harnessed to control gene expression.
A wealth of RNAs or RNA motifs are instrumental in controlling a variety of post-transcriptional or post-translational regulations. In this regard, selenocysteine incorporation in response to a redefined UGA stop codon certainly constitutes an intriguing and fascinating process. Translation elongation factors specialized for selenocysteine are needed to decode UGA selenocysteine codons. Discrimination between UGA selenocysteine and UGA stop codons also necessitates selenoprotein mRNA hairpins, called SECIS, that are internal to the coding frame in eubacteria or contained in the 3' untranslated regions in archaea/eukaryotes. This dichotomy leads to SECIS RNAs with distinct sequences and structures that tether the specialized translation elongation factor in a direct or indirect fashion, depending on the location of the SECIS RNA. The scope of this review is to bring a sharper focus on the SECIS RNA structures and SECIS RNA-protein complexes involved. Obviously, the examples described here highlight once again the versatility in form and function of RNA.
Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well.
Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria. Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine. We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal. In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds. We also show that SAM directly binds to and induces a conformational change in the leader RNA. Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo. Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo. These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.
Genetic control by metabolite-binding mRNAs is widespread in prokaryotes. These riboswitches are typically located in noncoding regions of mRNA, where they selectively bind their target compound and subsequently modulate gene expression. We have identified mRNA elements in fungi and in plants that match the consensus sequence and structure of thiamine pyrophosphate-binding domains of prokaryotes. In Arabidopsis, the consensus motif resides in the 3'-UTR of a thiamine biosynthetic gene, and the isolated RNA domain binds the corresponding coenzyme in vitro. These results suggest that metabolite-binding mRNAs are possibly involved in eukaryotic gene regulation and that some riboswitches might be representatives of an ancient form of genetic control.
Riboswitches are metabolite binding domains within certain messenger RNAs that serve as precision sensors for their corresponding targets. Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression. We have identified a class of riboswitches that selectively recognizes guanine and becomes saturated at concentrations as low as 5 nM. In Bacillus subtilis, this mRNA motif is located on at least five separate transcriptional units that together encode 17 genes that are mostly involved in purine transport and purine nucleotide biosynthesis. Our findings provide further examples of mRNAs that sense metabolites and that control gene expression without the need for protein factors. Furthermore, it is now apparent that riboswitches contribute to the regulation of numerous fundamental metabolic pathways in certain bacteria.
Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.
Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.
Metabolite-sensing mRNAs: Riboswitches are highly structured RNA domains that typically reside in the 5′-untranslated regions of certain bacterial mRNAs. These RNA domains serve as ligand-dependent genetic switches that provide a means by which RNA can control gene expression without the need for protein factors. Distinct classes of riboswitches are responsive to seven different metabolites and guide the maintenance of fundamental biochemical processes. One example is the regulation of the E. coli thiM mRNA by thiamine pyrophosphate (TPP; see representation).