The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 Å resolution (original) (raw)
Related papers
Base-Pairing between 23S rRNA and tRNA in the Ribosomal A Site
Molecular Cell, 1999
protected by P site-bound tRNA (Moazed and Noller, Baltimore, Maryland 21205 1989). One of these nucleotide protections led to the identification of a Watson-Crick base-pairing interaction between G2252 in domain V of 23S rRNA (a universally Summary conserved nucleotide) (Samaha et al., 1995) and C74 of P site bound tRNA. Interactions between other conserved The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-pnucleotides of domain V and the universal A76 and C75 puromycin (s 4 TCPm) photochemically cross-links with of peptidyl tRNA remain elusive (Samaha et al., 1995; high efficiency and specificity to G2553 of 23S rRNA Spahn et al., 1996b; Green et al., 1997; Saarma et al., and is peptidyl transferase reactive in its cross-linked 1998) although a Hoogsteen pairing interaction between state, establishing proximity between the highly con-U2585 and A76 has been proposed (Porse et al., 1996). served 2555 loop in domain V of 23S rRNA and the In the A site, tRNA footprinting analysis similarly idenuniversally conserved CCA end of tRNA. To test for tified nucleotides in domain V of 23S rRNA that were base-pairing interactions between 23S rRNA and aminoprotected by A site-bound tRNA and that might thereacyl tRNA, site-directed mutations were made at the fore be involved in direct rRNA-tRNA interactions; these universally conserved nucleotides U2552 and G2553 positions included both G2553 and U2555 in the highly of 23S rRNA in both E. coli and B. stearothermophilus conserved 2555 loop (Moazed and Noller, 1989). Reribosomal RNA and incorporated into ribosomes. Mucently, we identified a highly specific and efficient crosstations at G2553 resulted in dominant growth defects in link between a photoactivated aminoacyl tRNA analog, E. coli and in decreased levels of peptidyl transferase 4-thio-dT-p-C-p-Puromycin (s 4 TCPm), and G2553 of activity in vitro. Genetic analysis in vitro of U2552 and 23S rRNA (Green et al., 1998); this cross-linked s 4 TCPm G2553 mutant ribosomes and CCA end mutant tRNA substrate is highly reactive in the peptidyl transferase substrates identified a base-pairing interaction bereaction. These data place the CCA end of A site tRNA tween C75 of aminoacyl tRNA and G2553 of 23S rRNA. in the vicinity of the 2555 loop of domain V of 23S rRNA (Figure 1). Two additional experiments place the 2555 loop near the CCA end of the A site tRNA: genetic studies * To whom correspondence should be addressed (e-mail: ragreen@ jhmi.edu). previously identified P site interaction (Samaha et al., Lieberman, K.R., and Dahlberg, A.E. (1995). Ribosome-catalyzed peptide-bond formation. Prog. Nucleic Acid Res. Mol. Biol. 50, 1-23. Thanks to H. Noller for the work initiated in his laboratory; to B. Merryman, C., Moazed, D., McWhirter, J., and Noller, H.F. (1999a). Cormack, K. Lieberman, and C. Greider for critical reading of the Nucleotides in 16S rRNA protected by the association of 30S and manuscript; to G. Culver for Figure 1B; to J. Puglisi, E. Blackburn, 50S ribosomal subunits. J. Mol. Biol. 285, 97-105. . rRNA in the ribosomal A, P, and E sites. Cell 57, 585-597. Monro, R.E., and Marcker, K.A. (1967). Ribosome-catalysed reaction of puromycin with a formylmethionine-containing oligonucleotide. References J. Mol. Biol. 25, 347-350. Newman, A.J., and Norman, C. (1992). U5 snRNA interacts with exon Ban, N., Nissen, P., Hansen, J., Capel, M., Moore, P.B., and Steitz, sequences at 5Ј and 3Ј splice sites. Cell 68, 743-754. T.A. (1999). Placement of protein and RNA structures into the 5 Å -resolution map of the 50S ribosomal subunit. Nature 400, 841-847. Noller, H.F., and Woese, C.R. (1981). Secondary structure of 16S ribosomal RNA. Science 212, 403-411. Bhuta, P., Kumar, G., and Chladek, S. (1982). The peptidyltransferase center of Escherichia coli ribosomes: binding sites for the cytidine Noller, H.F., Kop, J., Wheaton, V., Brosius, J., Gutell, R.R., Kopylov, 3Ј-phosphate residues of the aminoacyl-tRNA 3Ј-terminus and the A.M., Dohme, F., Herr, W., Stahl, D.A., Gupta, R., and Waese, C.R. interrelationships between the acceptor and donor sites. Biochim. (1981). Secondary structure model for 23S ribosomal RNA. Nucleic Acids Res. 9, 6167-6189. Biophys. Acta 696, 208-211.
Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunits
Journal of Molecular Biology, 1999
We have studied the effect of subunit association on the accessibility of nucleotides in 23 S and 5 S rRNA. Escherichia coli 50 S subunits and 70 S ribosomes were subjected to a combination of chemical probes and the sites of attack identi®ed by primer extension. Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions. Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23 S rRNA; no changes are seen in 5 S rRNA. Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50 S subunit upon association with the 30 S subunit. Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920. In free 50 S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50 S subunit. When the 30 S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stemloops are protected, as are many bases in and around the corresponding apical loops. Intriguingly, three of the protected regions of 23 S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center. Together with the juxtaposition of the subunit-protected regions of 16 S rRNA with the small subunit tRNA binding sites, our ®ndings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30 S subunit with the peptidyl transferase center of the 50 S subunit via rRNA-rRNA interactions.
Journal of Molecular Biology, 1998
Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A threedimensional reconstruction at 21 A Ê resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site ®nger in the large subunit) could all be clearly identi®ed in the 80 S particle. On the other hand, signi®cant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into ®ve groups. The ®rst (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the ®fth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.
Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome
Biochimie, 2013
Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA Asp analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3 0-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.
Arrangement of 3′-terminus of tRNA on the human ribosome as revealed from cross-linking data
Biochimie, 2008
This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3 0 -end of tRNA in the 80S ribosomal A and P sites using a tRNA Asp analogue that bears a 4-thiouridine (s 4 U) attached to the 3 0 -terminal adenosine. It was shown that an additional nucleotide s 4 U77 on the 3 0 -end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302e4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides crosslinked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s 4 U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3 0 -terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.
Nucleic Acids Research, 1981
We determined 90% of the primary structure of E.coli MRE 600 23S rRNA by applying the sequencing gel technique to products of Tl, SI, A and Naja oxiana nuclease digestion. Eight cistron heterogeneities were detected, as well as 16 differences with the published sequence of a 23S rRNA gene of an E.coli K12 strain. The positions of 13 post-transcriptionally modified nucleotides and of single-stranded, double-stranded and subunit surface regions of E.coli 23S rRNA were identified. Using these experimental results and by comparing the sequences of E.coli 23S rRNA, maize chloro. 23S rRNA and mouse and human mit 16S rRNAs, we built models of secondary structure for the two 23S rRNAs and for large portions of the two mit rRNAs. The structures proposed for maize chloroplast and E.coli 23S rRNAs are very similar, consisting of 7 domains closed by long-range base-pairings. In the mitochondrial 16S rRNAs, 3 of these domains are strongly reduced in size and have a very different primary structure compared to those of the 23S rRNAs. These domains were previously found to constitute a compact area in the E.coli 50S subunits. The conserved domains do not belong to this area and contain almost all the modified nucleotides. The most highly conserved domain, 2042-2625, is probably part of the ribosomal A site. Finally, our study strongly suggests that in cytoplasmic ribosomes the 3'-end of 5.8S rRNA is basepaired with the 5'-end of 26S or 28S rRNA. This confirms the idea that 5.8S RNA is the counterpart of the 5'-terminal region of prokaryotic 23S rRNA.
FEBS Letters, 2011
Helix 89 of the 23S rRNA connects ribosomal peptidyltransferase center and elongation factor binding site. Secondary structure of helix 89 determined by X-ray structural analysis involves less base pairs then could be drawn for the helix of the same primary structure. It can be that alternative secondary structure might be realized at some stage of translation. Here by means of site-directed mutagenesis we stabilized either the ''X-ray'' structure or the structure with largest number of paired nucleotides. Mutation UU2492-3C which aimed to provide maximal pairing of the helix 89 of the 23S rRNA was lethal. Mutant ribosomes were unable to catalyze peptide transfer independently either with aminoacyl-tRNA or puromycin.
Nucleic Acids Research, 1999
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 [ 32 P]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.
Contacts between the growing peptide chain and the 23S RNA in the 50S ribosomal subunit
Nucleic Acids Research, 1994
Peptides of defined length carrying a diazirine photoaffinity label attached either to the a-NH 2 group of the N-terminal methionine residue, or to the e-NH 2 group of an immediately adjacent lysine residue, were prepared in situ on Escherichia coli ribosomes in the presence of a synthetic mRNA analogue. Peptide growth was stopped simply by withholding the aminoacyl-tRNA cognate to an appropriate downstream codon. After photo-activation at 350 nm the sites of cross-linking to ribosomal RNA were determined by our standard procedures; the C-terminal amino acid of each peptide was labelled with tritium, in order to confirm whether the individual cross-linked complexes contained the expected 'full-length' peptide, as opposed to shorter products. The shortest peptides became cross-linked to sites within the 'peptidyl transferase ring' of the 23S RNA, namely to positions 2062, 2506, 2585 and 2609. However, already when the peptide was three or four residues long, a new crosslink was observed several hundred nucleotides away in another secondary structural domain; this site, at position 1781, lies within one of several RNA regions which have been implicated in other studies as being located close to the peptidyl transferase ring. Further application of this approach, combined with modelbuilding studies, should enable the path of the nascent peptide through the large ribosomal subunit to be definitively mapped. MATERIALS AND METHODS Preparation of mRNA, and tRNA derivatives An mRNA analogue was prepared by T7 transcription from a synthetic DNA template (9,11). This mRNA had the sequence GGG AGA AAG AAA AUG AAA UUC GAA CUG GAC ACC, carrying codons for methionine, lysine, phenylalanine and glutamic acid (M, K, F, E, underlined). Individual tRNA species