Figure 2 - uploaded by Phil Mitchell
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Ribonuclease T1 fingerprints of a non-cross-linked RNA fragment (panels A and B) and the corresponding cross-linked complex (panels C and D) from positions ca. 1911-2054 in the 23S RNA sequence. Panels A and C show the 'standard' chromatographic system, panels B and D the 'alternative' system (see Materials and Methods). Direction of chromatography is from right to left in the first dimension and from bottom to top in the second dimension. Sample application points in panels B and D are denoted by '+'. The asterisks at the sample application points in panels A and C indicate oligonucleotides which resolve into pairs of spots (also asterisked) in panels B and D, respectively. Oligonucleotides 1, 2 and 3 from the non-cross-linked fragment (indicated by arrows in panels A and
Source publication
Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coli by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were
then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleoti...
Contexts in source publication
Context 1
... such as strain or cistronic heterogeneities, or sites of nucleoside or sugar methylations. In conjunction with the use of the 'alternative' chromatographic elution system (see Materials and Methods and ref. (19)), this comparison allows a direct unequivocal assignment of the sites involved in cross-links. An example of this method is given in Fig. 2, which shows 'standard' and 'alternative' ribonuclease T, fingerprints of the cross-linked complex 5 (see AAATp spot in the secondary analysis. 5: AAATUCCUUGp, ACCUGp and CACGp were absent from the fibonuclease T, fingerprint analyses (see Fig. 2 Table 1 below) and the corresponding non-cross-linked RNA fragment between positions ca. ...
Context 2
... unequivocal assignment of the sites involved in cross-links. An example of this method is given in Fig. 2, which shows 'standard' and 'alternative' ribonuclease T, fingerprints of the cross-linked complex 5 (see AAATp spot in the secondary analysis. 5: AAATUCCUUGp, ACCUGp and CACGp were absent from the fibonuclease T, fingerprint analyses (see Fig. 2 Table 1 below) and the corresponding non-cross-linked RNA fragment between positions ca. 1911-2054 of the 23S RNA sequence. Three oligonucleotides which are present in the control fragment (indicated by arrows) but which are absent from the cross-linked complex, constitute the cross-linked ribonuclease T1 residue. This gives an ...
Context 3
... which were isolated by ribonuclease H digests using sets of deoxyoligonucleotides complementary to sites along the whole length of the 23S RNA sequence (cf. ref. 19); these were reported in our earlier studies, but have now been more precisely localised by oligonucleotide fingerprinting using the 'alternative' chromatographic elution system (cf. Fig. 2) and, in the case of cross-link 2, ribonuclease U2 digestion of the cross-linked ribonuclease T, residue. Of the seven new cross-links, six represent tertiary contacts in the folding of the rRNA and thus can be used to impose spatial constraints on the phylogenetically established secondary structure of 23S RNA in model building ...
Similar publications
The sarcin-ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analy...
Citations
... Model of the 23s rRNA structure is adapted from Mitchell et al. (1990). tRNA -23s rRNA cross-links are examined in the context of the three-dimensional model of 23s rRNA developed by Brimacombe and co-workers (Mitchell et al. 1990). As shown inFig. ...
The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3' end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.
... Figure 1 also shows interRNA and intraRNA cross-links involving the same areas of the ribosomal RNA molecules that have just been discussed. These data include intraRNA crosslinks near to the 3' end of the 16s RNA (Fig. 1, e; Doring et al. 1992), an intraRNA cross-link in the central region of the 16s RNA ( Fig. 1, g; Atmadja et al. 1986), interRNA cross-links connecting the 16s and 23s molecules (Fig. 1, h; , intraRNA cross-links connecting two different domains of the 23s RNA (Fig. 1, j, Mitchell et al. 1990;Dijring et al. 1991), and an interRNA cross-link connecting the 5 s and 23s molecules (Fig. 1, o; Dontsova et al. 1994). ...
... This cross-link is reproducibly specific for tRNA bound to the A-site and lies at a position (not yet finally localized) approximately between nucleotides 875 and 905. The result is surprising because the long helix 38 has so far been virtually devoid of any topographical information (see, e.g., Brimacombe 1995), and in our preliminary three-dimensional model for the 23s RNA (Mitchell et al. 1990), the helix was located at the solvent side of the 50s subunit, far away from the ribosomal A site. The second example (Fig. 2, bottom) consists of two cross-links from position 47 of ~R N A '~~ located at the P-site. ...
Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli. The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA. In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA. The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice. These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible. The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models.
... The main disadvantage is that in larger RNAs the site of crosslinking can be distant from the labeled end of the molecule (>150–200 nts) and thus more difficult to resolve on standard sequencing gels. Alternative, more complex, yet very accurate methods, however, have been developed in the mapping of crosslinks in ribosomal RNA of using RNase H and RNA fingerprinting (Mitchell et al., 1990). ...
RNA-RNA crosslinking provides a rapid means of obtaining evidence for the proximity of functional groups in structurally complex RNAs and ribonucleoproteins. Such evidence can be used to provide a physical context for interpreting structural information from other biochemical and biophysical methods and for the design of further experiments. The identification of crosslinks that accurately reflect the native conformation of the RNA of interest is strongly dependent on the position of the crosslinking agent, the conditions of the crosslinking reaction, and the method for mapping the crosslink position. Here, we provide an overview of protocols and experimental considerations for RNA-RNA crosslinking with the most commonly used long- and short-range photoaffinity reagents. Specifically, we describe the merits and strategies for random and site-specific incorporation of these reagents into RNA, the crosslinking reaction and isolation of crosslinked products, the mapping crosslinked sites, and assessment of the crosslinking data.
... Lone-pair or tertiary interactions are base pairs that are not immediately adjacent to other base pairs (Larsen, 1992; Gutell & Damberger, 1996). The Gutell model describes a number of such interactions, supported by covariation analysis and in some cases by experimental evidence (e.g. Mitchell et al., 1990). In the Gutell model, three lone-pair interactions can be evaluated using the data presented here. ...
... The first interaction is between nucleotides 2282 and 2427 of the Escherichia coli sequence. We find no support in the form of covarying substitutions in insect mtDNA for this proposed interaction despite the fact that substitutions are common in this region and the interaction is supported by crosslinking experiments (Mitchell et al., 1990). This interaction is unable to form in many of the tiger beetles (Vogler & Pearson, 1996 ), odonates (unpublished data) and cockroaches (Kambhampati, 1995) for example. ...
We have analysed over 400 partial insect mitochondrial large subunit (mit LSU) sequences in order to identify conserved motifs and secondary structures for domains IV and V of this gene. Most of the secondary structure elements described by R. R. Gutell et al. (unpublished) for the LSU were identified. However, we present structures for helices 84 and 91 that are not recognized in previous universal models. The portion of the 16S gene containing domains IV and V is frequently sequenced in insect molecular systematic studies so we have many more sequences than previous studies which focused on the complete mitochondrial LSU molecule. In addition, we have the advantage of investigating several sets of closely related taxa. Aligned sequences from thirteen insect orders and nine secondary structure diagrams are presented. These conserved sequence motifs and their associated secondary structure elements can now be used to facilitate the alignment of other insect mit LSU sequences.
... Fe(II) tethered to L9 targeted speci®c regions of domains I, III, IV, and V in the 23 S rRNA secondary structure (Figure 5(a)-(e)). These data reinforce the emerging picture, drawn from rRNA-rRNA crosslinking studies (Mitchell et al., 1990 ), phylogenetic covariation analysis (Gutell & Woese, 1990), directed hydroxyl radical probing experiments using ribosomal proteins L11 (Holmberg & Noller, 1999) and L15 (Lieberman & Noller, 1998), and X-ray crystallographic analysis (Ban et al., 1998Ban et al., , 1999 Cate et al., 1999 ) of a 50 S subunit architecture dominated by interwoven, interdomain interactions. Such a structural organization is in marked contrast to that observed for the 30 S ribosomal subunit, where individual secondary structural domains largely comprise autonomous morphological domains (Clemons et al., 1999). ...
Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.
... Most of the available data from these approaches have been obtained with ribosomes from the eubacterium Escherichia coli, and a number of research groups (e.g. Brimacombe et al., 1988; Nagano et al., 1988; Stern et al., 1988; Mitchell et al., 1990; Malhotra & Harvey, 1994) have made use of different combinations of the data sets to derive 3D models for the E. coli 16 S or 23 S rRNA molecules. However, these models are essentially only``only``cartoons'' of the ribosome, and it is abundantly clear that on its own this type of molecular modelling approach could never lead to a structure of the ribosome at atomic resolution. ...
... We adopted the following strategy. A ``wire-and-tube'' model, previously used to construct a preliminary 3D model of the 23 S rRNA (Mitchell et al., 1990), was taken as the starting point, and this was adapted so as to incorporate the more recent biochemical information (Figure 1). (With large molecules such as the 23 S rRNA, ``physical'' wire-and-tube models still have certain advantages over computer-generated models, in particular for gaining an overview of the complete structure.) ...
The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A. Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post-transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure.
... This conclusion was in agreement with the observation that 5 S rRNA is largely inaccessible in the ribosome (Farber & Cantor, 1981; Noller & Herr, 1974; Silberklang et al., 1983 ) making its participation in any ribosomal activity involving direct interaction with ribosomal ligands highly unlikely. Immunoelectron microscopy placed 5 S rRNA in the central protuberance of 50 S subunits, not far from the proposed location of the ribosomal peptidyl transferase (Shatsky et al., 1980; Sto È ef¯er & Sto È ef¯er-Melicke, 1986; Mitchell et al., 1990). The proximity of 5 S rRNA to the ribosome catalytic center is consistent with the observation that large ribosomal subunits assembled without 5 S rRNA are signi®cantly compromised in their peptidyl transferase activity (Silberklang et al., 1983). ...
Functional large ribosomal subunits of Thermus aquaticus can be reconstituted from ribosomal proteins and either natural or in vitro transcribed 23 S and 5 S rRNA. Omission of 5 S rRNA during subunit reconstitution results in dramatic decrease of the peptidyl transferase activity of the assembled subunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstitution can partly restore the activity of ribosomal subunits assembled without 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most active: activity of the subunits assembled in the absence of 5 S rRNA was increased more than 30-fold if antibiotic was present during reconstitution procedure. Activity of the subunits assembled with 5 S rRNA was also slightly stimulated by RU69874, but to a much lesser extent, approximately 1.5-fold. Activity of the native T. aquaticus 50 S subunits incubated in the reconstitution conditions in the presence of RU69874 was, in contrast, slightly decreased. The presence of antibiotics was essential during the last incubation step of the in vitro assembly, indicating that drugs affect one of the last assembly steps.
... A 23S rRNA region comprising helices 82-87 was identified many years ago (33) in a ribonucleoprotein fragment consisting of 5S rRNA, proteins L5, L18 and L25 (the 5S-associated proteins; 7-10) and a part of the 23S molecule. The same components clearly form the central protuberance of the 50S subunit (34). RNA-protein cross-links to proteins L5 and L18 have been observed in helix 84, as well as cross-links to L27 in helices 81 and 85 (35); L27 is known from IEM studies (5) to lie at the base of the central protuberance. ...
Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33–71 of
the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions
40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p-azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S
ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation
at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple crosslinks from 5S rRNA to two distinct
regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links
at sites between nucleotides 885 and 922. The second region covered helices 81–85 in Domain V, with sites between nucleotides
2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative
to the 23S rRNA within the 50S subunit.
... The N7 position of the adjacent nucleotide G748 also became weakly reactive to DMS. The 750 loop, consisting of the sequence 5 H m 1 GÉm 5 UGAAAA-3 H , has been crosslinked via m 5 U747 to the vicinity of U2613 (Mitchell et al., 1990). This position is adjacent to the central loop of domain V and in close proximity to A2058 and other nucleotides implicated in macrolide antibiotic interactions. ...
We have used chemical modification to examine the conformation of 23 S rRNA in Escherichia coli ribosomes bearing erythromycin resistance mutations in ribosomal proteins L22 and L4. Changes in reactivity to chemical probes were observed at several nucleotide positions scattered throughout 23 S rRNA. The L4 mutation affects the reactivity of G799 and U1255 in domain II and that of A2572 in domain V. The L22 mutation influences modification in domain II at positions m5U747, G748, and A1268, as well as at A1614 in domain III and G2351 in domain V. The reactivity of A789 is weakly enhanced by both the L22 and L4 mutations. None of these nucleotide positions has previously been associated with macrolide antibiotic resistance. Interestingly, neither of the ribosomal protein mutations produces any detectable effects at or within the vicinity of A2058 in domain V, the site most frequently shown to confer macrolide resistance when altered by methylation or mutation. Thus, while L22 and L4 bind primarily to domain I of 23 S rRNA, erythromycin resistance mutations in these ribosomal proteins perturb the conformation of residues in domains II, III and V and affect the action of antibiotics known to interact with nucleotide residues in the peptidyl transferase center of domain V. These results support the hypothesis that ribosomal proteins interact with rRNA at multiple sites to establish its functionally active three-dimensional structure, and suggest that these antibiotic resistance mutations act by perturbing the conformation of rRNA.
... As regards its 23 S rRNA environment, L6 has been cross-linked to the end of a stem-loop structure (nucleotides 2455 to 2496 in Escherichia coli) that projects from domain V (Wower et al., 1981). Domain V is a highly conserved region of the 23 S molecule that has been implicated in peptide bond formation (Vester & Garrett, 1988) and the binding of tRNA at the A and P sites (Steiner et al., 1988; Mitchell et al., 1990; Brimacombe et al., 1993). L6 also binds to a fragment of 23 S rRNA corresponding to domain VI (Leffers et al., 1988 ). ...
... Second, L6 is close to the GTPase center in the region of the L7/L12 stalk (Walleczek et al., 1988; Spirin & Vasiliev, 1989), and can be cross-linked to EF-G (Sko È ld, 1982), which binds in the same location as EF-Tu. Third, L6 has been associated with domains V and VI of 23S rRNA (Wower et al., 1981; Leffers et al., 1988), both of which have been implicated in the binding of the EF-Tu Á GTP Áaminoacyl tRNA ternary complex (Leffers et al., 1988; Moazed et al., 1988; Moazed & Noller, 1989; Mitchell et al., 1990). The only other 50 S component in which mutations affect proofreading is the a-sarcin loop. ...
Antibiotic resistance is rapidly becoming a major medical problem. Many antibiotics are directed against bacterial ribosomes, and mutations within both the RNA and protein components can render them ineffective. It is well known that the majority of these antibiotics act by binding to the ribosomal RNA, and it is of interest to understand how mutations in the ribosomal proteins can produce resistance. Translational accuracy is one important target of antibiotics, and a number of ribosomal protein mutations in Escherichia coli are known to modulate the proofreading mechanism of the ribosome. Here we describe the high-resolution structures of two such ribosomal proteins and characterize these mutations. The S5 protein, from the small ribosomal unit, is associated with two types of mutations: those that reduce translational fidelity and others that produce resistance to the antibiotic spectinomycin. The L6 protein, from the large subunit, has mutations that cause resistance to several aminoglycoside antibiotics, notably gentamicin. In both proteins, the mutations occur within their putative RNA-binding sites. The L6 mutations are particularly drastic because they result in large deletions of an RNA-binding region. These results support the hypothesis that the mutations create local distortions of the catalytic RNA component.When combined with a variety of structural and biochemical data, these mutations also become important probes of the architecture and function of the translational machinery. We propose that the C-terminal half of S5, which contains the accuracy mutations, organizes RNA structures associated with the decoding region, and the N-terminal half, which contains the spectinomycin-resistance mutations, directly interacts with an RNA helix that binds this antibiotic. As regards L6, we suggest that the mutations indirectly affect proofreading by locally distorting the EF-Tu.GTP.aminoacyl tRNA binding site on the large subunit.
