Rapid ribosomal translocation depends on the conserved 18-55 base pair in P-site transfer RNA

Pan, Dongli; Kirillov, S.V.; Zhang, Chunmei; Hou, Ya‐Ming; Cooperman, Barry S.

doi:10.1038/nsmb1074

Cited by 47 publications

(62 citation statements)

References 36 publications

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“…Subsequent recognition of the start codon allows P i release to occur, making hydrolysis of GTP irreversible. The mechanism of EF-G-dependent translocation is similar in that P i release occurs much more slowly than GTP hydrolysis (12,26) and is limited by a conformational rearrangement (12,40). Further experiments will be required to determine the internal equilibrium position of GTP and GDP⅐P i within ribosome-bound EF-G.…”

Section: Discussionmentioning

confidence: 99%

“…The rate of single-turnover GTP hydrolysis was determined essentially as described in ref. 26. To form PRE complexes, control or mutant ribosomes (2 M) were first incubated with message m404 (4 M; see ref.…”

Section: Methodsmentioning

confidence: 99%

“…To form EF-G⅐GTP, EF-G (3 M) was incubated with [␥-32 P]GTP (Perkin-Elmer; 50 M, Ϸ1,000 dpm/pmol) in buffer 3 for 2 min at 37°C and then placed on ice. Equal volumes of PRE complex (1 M final) and EF-G⅐GTP (1.5 M final) were then rapidly mixed at 25°C in a quench-flow apparatus (Kintek) for indicated times before quenching with 0.6 M HClO 4 containing 1.8 mM KH 2PO4, as described (26). Data were fit to the equation y ϭ A⅐exp(Ϫk app1⅐t) ϩ kapp2⅐t ϩ C; where A is the burst amplitude, kapp1 is the apparent burst rate, t is time, k app2 is the apparent turnover rate, and C is a constant.…”

Section: Methodsmentioning

confidence: 99%

“…To measure the rate of single-turnover GTP hydrolysis, we used chemical quench flow as described in ref. 26. PRE complexes were assembled with control or C2394A ribosomes and then mixed with EF-G⅐[␥-32 P]GTP for various periods of time before quenching with 0.6 M perchloric acid.…”

Section: Mutation C2394a Does Not Decrease the Rate Of Ef-g-dependentmentioning

confidence: 99%

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Role of hybrid tRNA-binding states in ribosomal translocation

Walker

Shoji

Pan

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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108

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During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steadystate kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k trans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K 1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K 1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k trans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.elongation factor G ͉ translation ͉ exit site ͉ GTPase M ovement of tRNAs through the ribosome is believed to occur in a stepwise manner (1). Chemical protection experiments showed that, after peptidyl transfer, the acceptor ends of the tRNAs can move spontaneously with respect to the 50S subunit to form the hybrid state. In the hybrid state, the deacylated tRNA occupies the 30S P site and 50S E site (P/E site) while the peptidyl-tRNA occupies the 30S A site and 50S P site (A/P site). It was proposed that hybrid state formation precedes codon-anticodon movement within the 30S subunit, and only the latter event requires catalysis by elongation factor G (EF-G). Support for the hybrid-state model came from Förster resonance energy transfer (FRET) experiments in which an Ϸ20-Å movement of the 5Ј end of the newly deacylated tRNA toward ribosomal protein L1 was inferred after peptide bond formation, whereas the position of the peptidyl group changed little (2). Because L1 lies near the 50S E site, these data are consistent with movement of tRNA into the P/E site. Further evidence came from single-molecule FRET studies that monitored the distance between probes attached to the elbows of tRNAs in the A and P sites. Fluctuations between two distinct configurations were observed that were structurally consistent with the classical and hybrid states (3). More recently, a third state of the pretranslocation (PRE) complex was observed by single-molecule FRET, consistent with one tRNA bound to the P/E site and the other bound to the A/A site (4). Experiments that monitored mRNA in ribos...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mutation C2394a Does Not Decrease the Rate Of Ef-g-dependentmentioning

confidence: 99%

See 2 more Smart Citations

Role of hybrid tRNA-binding states in ribosomal translocation

Walker

Shoji

Pan

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

108

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“…Large facilities and the ribosome A. Yonath complex D50S-ASM . Although the PTC has significant tolerance in the positioning of fragment reaction substrates (Yonath 2003), the interactions of the tRNA acceptor stem seem to be crucial for accurate substrate positioning in the PTC at the configuration allowing for peptide bond formation, in accord with the finding that the tRNA core region is functionally important for its dynamic interactions with the ribosome (Pan et al 2006). The linkage between the elaborate architecture of the symmetrical region and the position of the A-site tRNA indicates that the translocation of the tRNA 3 0 end is performed by a combination of independent, albeit synchronized motions: a sideways shift, performed as a part of the overall mRNA/tRNA translocation, and a rotatory motion of the A-tRNA 3 0 end along a path confined by the PTC walls.…”

Section: Motions Within the Peptidyl Transferase Centrementioning

confidence: 69%

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

Yonath

2009

J. R. Soc. Interface.

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Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric prebiotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

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Winterschlafende Bären, Antibiotika und die Evolution des Ribosoms (Nobel‐Aufsatz)

Yonath

2010

Angewandte Chemie

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Dank der hochaufgelösten Strukturen von Ribosomen, der zellulären Maschinen, die den genetischen Code in Proteine übersetzen, konnten zahlreiche ribosomale Aktivitäten und Strukturdetails aufgedeckt und erklärt werden: der Mechanismus der DNA‐Decodierung, der Prozessierungspfad der mRNA, die Bindungsstellen der tRNA im Ribosom, die Position und Eigenschaften des ribosomalen Austrittstunnels der naszierenden Proteine und die Wechselwirkungen des Ribosoms mit nicht‐ribosomalen Faktoren wie den Initiations‐, Freisetzungs‐ und Recyclingfaktoren. Vor allem haben diese Strukturen den Beweis erbracht, dass das Ribosom ein Ribozym ist, dessen aktives Zentrum – d. h. die Stelle, an der die Bildung der Peptidbindungen erfolgt – in einer symmetrischen Region liegt, die in die ansonsten unsymmetrische Ribosomstruktur eingebettet ist. Diese symmetrische Region ist hoch konserviert und enthält bereits alle Komponenten, die für die Bildung der Peptidbindungen und die Polymeraseaktivität des Ribosoms erforderlich sind. Sie könnte daher das Relikt eines Protoribosoms sein, einer dimeren präbiotischen Funktionseinheit, die Peptidbindungen und uncodierte Polypeptidketten bildete. Strukturen von Ribosom‐Antibiotika‐Komplexen haben die Grundlagen für eine klinische Verwendung dieser Antiobiotika geschaffen, Resistenzmechanismen aufgedeckt und Aufschluss darüber gegeben, wie Antibiotika zwischen pathogenen Bakterien und Wirtzellen differenzieren. Damit haben diese Forschungen wertvolle Strukturinformationen für die Verbesserung von Antibiotika und den Entwurf neuartiger Verbindungen, die als Antibiotika dienen könnten, geliefert.

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Rapid ribosomal translocation depends on the conserved 18-55 base pair in P-site transfer RNA

Cited by 47 publications

References 36 publications

Role of hybrid tRNA-binding states in ribosomal translocation

Role of hybrid tRNA-binding states in ribosomal translocation

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

Winterschlafende Bären, Antibiotika und die Evolution des Ribosoms (Nobel‐Aufsatz)

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