Translocation of tRNA during protein synthesis

Noller, Harry F.; Yusupov, Marat; Yusupova, G.; Baucom, Albion; Cate, J.H.D.

doi:10.1016/s0014-5793(02)02327-x

Cited by 133 publications

(95 citation statements)

References 40 publications

(63 reference statements)

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“…The results are the means of at least four independent experiments with bars representing S.D. (69) and requires a sliding of the mRNA together with the tRNA (70,71). We hypothesize that translocation requires disruption of the S7-S11 interaction, releasing the grip on the mRNA while allowing a movement of the head relative to the platform.…”

Section: Discussionmentioning

confidence: 99%

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

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In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.The ribosome is the cellular machinery responsible for protein synthesis in all living organisms. The elucidation of the crystal structure of the prokaryotic ribosome has led to a major progress in understanding its function (1-7). Analysis of the ribosome structure combined with a wealth of biochemical data clearly showed that ribosomal RNA (rRNA) is the key player in the functions of the ribosome, and it is currently assumed that the main task of the ribosomal proteins is to help and stabilize rRNA folding and to facilitate conformational changes in rRNA (8 -10). However, a growing list of examples suggests that ribosomal proteins directly participate to protein synthesis. The x-ray structure of the ribosome reveals that S12 is part of the decoding site (Refs. 11 and 12, reviewed in Ref. 13). It also shows that the 30 S proteins S13, S15, and the 50S proteins L2, L5, L14, L19 are involved in intersubunit bridges, while the 30 S proteins S9, S13, and the 50 S proteins L1, L5, L33 interact with tRNAs, suggesting that they could participate in the translocation step (12). Studies in solution also point to a role for the ribosomal proteins, such as an involvement in mRNA binding for S1 (14 -16), a participation in peptidyl transferase activity for L2 (17-19) and the prevention of mRNA slippage for L9 (20).Several protein-protein interactions were identified in the crystal structure of the 30 S subunit (21, 22) but, so far, only the interaction between proteins S4 and S5 has been directly related to a particular ribosome function, that is the control of translational fidelity. Indeed, mutations that disrupt the S4-S5 interaction decrease translational accuracy (23,24). Among the other protein-protein inter...

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Section: Discussionmentioning

confidence: 99%

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

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“…This motion is part of the translocation event, a fundamental act of the elongation cycle that could be performed by a straightforward shift, [69][70][71] or by incorporating intermediate hybrid states. 72 Within the PTC cavity, the rotatory motion of the tRNA 3 0 ends is likely to be propelled by A2602, an action connected to the shifts of the tRNA helical stems, which seem to be performed by the intersubunit bridge B2a, functioning as a molecular platform. A2602 and U2585, two universal nucleotides positioned in the middle of the PTC pattern, near the two-fold rotation axis, anchor the rotatory motion of the tRNA 3 0 end.…”

Section: Discussionmentioning

confidence: 99%

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

Zarivach

Bashan

Berisio

et al. 2004

J of Physical Organic Chem

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High-resolution structures of both ribosomal subunits revealed that most stages of protein biosynthesis, including decoding of genetic information, are navigated and controlled by the elaborate ribosomal architecturaldesign. Remote interactions govern accurate substrate alignment within a flexible active-site pocket [peptidyl transferase center (PTC)], and spatial considerations, due mainly to a universal mobile nucleotide, U2585, ensure proper chirality by interfering with D-amino-acids incorporation. tRNA translocation involves two correlated motions: overall mRNA/tRNA (messenger and transfer RNA) shift, and a rotation of the tRNA single-stranded aminoacylated-3 0 end around the bond connecting it with the tRNA helical-regions. This bond coincides with an axis passing through a sizable symmetry-related region, identified around the PTC in all large-subunit crystal structures. Propelled by a bulged universal nucleotide, A2602, positioned at the two-fold symmetry axis, and guided by a ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry optimal for peptide-bond formation and in geometry ensuring nascent proteins entrance into their exit tunnel. Hence, confirming that ribosomes contribute positional rather than chemical catalysis, and that peptide bond formation is concurrent with A-to P-site tRNA passage. Connecting between the PTC, the decoding center, the tRNA entrance and exit points, the symmetry-related region can transfer intra-ribosomal signals between remote functional locations, guaranteeing smooth processivity of amino acids polymerization. Ribosomal proteins are involved in accurate substrate placement (L16), discrimination and signal transmission (L22) and protein biosynthesis regulation (CTC). Residing on the exit tunnel walls near its entrance, and stretching to its opening, protein-L22 can mediate ribosome response to cellular regulatory signals, since it can swing across the tunnel, causing gating and elongation arrest. Each of the protein CTC domains has a defined task. The N-terminal domain stabilizes the intersubunit-bridge confining the A-site-tRNA entrance. The middle domain protects the bridge conformation at elevated temperatures. The C-terminal domain can undergo substantial conformational rearrangements upon substrate binding, indicating CTC participation in biosynthesiscontrol under stressful conditions.

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“…Release of RRF from Model Post-termination ComplexesThe physiological substrate of RRF for the disassembly reaction is a ribosome complexed with mRNA (with a stop codon at the A-site) and deacylated tRNA(s) bound at the P/E sites (18,50). To establish that the release of RRF from vacant ribosomes by EF-G detailed above is indeed representative of part of the natural reaction catalyzed by RRF, the possible release of RRF from model post-termination complexes (7, 31) was examined.…”

Section: Binding Of Rrf To 70 S Ribosomes In the Presence Of Ef-g-mentioning

confidence: 99%

Release of Ribosome-bound Ribosome Recycling Factor by Elongation Factor G

Kiel

Raj

Kaji

et al. 2003

Journal of Biological Chemistry

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Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.

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Translocation of tRNA during protein synthesis

Cited by 133 publications

References 40 publications

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Functional aspects of ribosomal architecture: symmetry, chirality and regulation

Release of Ribosome-bound Ribosome Recycling Factor by Elongation Factor G

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