Author
Yuri P. Semenkov
Other affiliations: Russian Academy of Sciences, Max Planck Society
Bio: Yuri P. Semenkov is an academic researcher from Petersburg Nuclear Physics Institute. The author has contributed to research in topics: Transfer RNA & Ribosome. The author has an hindex of 12, co-authored 13 publications receiving 757 citations. Previous affiliations of Yuri P. Semenkov include Russian Academy of Sciences & Max Planck Society.
Topics: Transfer RNA, Ribosome, A-site, Elongation factor, P-site
Papers
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TL;DR: The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.
Abstract: The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the α-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.
179 citations
TL;DR: The data suggest that initial binding of tRNA to the A site is followed by a rate-limiting rearrangement of the anticodon loop or the ribosome decoding center that is favored by purines at position 37 and involves stronger stacking, additional Mg(2+) binding, and interactions with 16S rRNA.
Abstract: The anticodon loop of tRNA contains a number of conserved or semiconserved nucleotides. In most tRNAs, a highly modified purine is found at position 37 immediately 3′ to the anticodon. Here, we examined the role of the base at position 37 for tRNAPhe binding to the A site of Escherichia coli ribosomes. Affinities and rate constants of A-site binding of native yeast peptidyl-tRNAPhe with hypermodified G (wybutine), or of unmodified peptidyl-tRNAPhe transcripts with G, A, C, or U, at position 37 were measured. The data indicate that purines stabilize binding due to stronger stacking and additional interactions with the ribosome mediated by Mg2+ ions. Paromomycin, an antibiotic that binds to 16S rRNA in the decoding center, greatly stabilized tRNAs in the A site and abolished the Mg2+-dependence of binding. Comparison of binding enthalpies and entropies suggests that hypermodification of the base at position 37 does not affect stacking in the codon–anticodon complex, but rather decreases the entropic penalty for A-site binding. Substitution of purines with pyrimidines at position 37 increases the rates of tRNA binding to and dissociation from the A site. The data suggest that initial binding of tRNA to the A site is followed by a rate-limiting rearrangement of the anticodon loop or the ribosome decoding center that is favored by purines at position 37 and involves stronger stacking, additional Mg2+ binding, and interactions with 16S rRNA.
113 citations
TL;DR: Upon transpeptidylation, the 3′ end of aminoacyl-tRNA (aa-t RNA) in the ribosomal A site enters the A/P hybrid state and constitutes an important element of the translocation mechanism.
Abstract: Upon transpeptidylation, the 3′ end of aminoacyl-tRNA (aa-tRNA) in the ribosomal A site enters the A/P hybrid state. We report that transpeptidylation of Phe-tRNA to fMetPhe-tRNA on Escherichia coli ribosomes substantially lowers the kinetic stability of the ribosome–tRNA complex and decreases the affinity by 18.9 kJ mol−1. At the same time, the free energy of activation of elongation factor G dependent translocation decreases by 12.5 kJ mol−1, indicating that part of the free energy of transpeptidylation is used to drive translocation kinetically. Thus, the formation of the A/P hybrid state constitutes an important element of the translocation mechanism.
109 citations
TL;DR: It is shown that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA, which lends support to the diffusion model of tRNA movement during translocation.
Abstract: During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA–mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA–mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.
93 citations
TL;DR: The allosteric three-site model of elongation of ribosomal tRNA exit (E) site is considered untenable because the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.
Abstract: For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.
80 citations
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Alternatives1, John Innes Centre2, University of Bonn3, University of North Carolina at Chapel Hill4, University of Wisconsin-Madison5, University of Utah6, University of Southern California7, University of Edinburgh8, University of Warwick9, Harvard University10, University College Cork11, University of Queensland12, University of Hertfordshire13, University of Potsdam14, University of California, San Diego15, Goethe University Frankfurt16, University of California, San Francisco17, University of Delaware18, Uppsala University19, Medical University of Vienna20, J. Craig Venter Institute21, University of Hawaii at Manoa22, Leibniz Association23, University of Iowa24, University of Aberdeen25, Georgia Institute of Technology26, University of California, Berkeley27, University of Groningen28, Princeton University29, University of Marburg30, University of Illinois at Urbana–Champaign31, Saarland University32, Norwegian University of Life Sciences33, Massey University34, Toyama Prefectural University35, ETH Zurich36, University of Saskatchewan37, Rutgers University38, Scripps Research Institute39, University of Helsinki40, Texas A&M University41, National Institutes of Health42, Technical University of Berlin43, University of Otago44, University of Cambridge45, University of Alberta46, Michigan State University47, Hofstra University48
TL;DR: This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products.
1,560 citations
TL;DR: The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (t RNA) and metal ions also stabilize the intersubunit interface.
Abstract: The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.
1,312 citations
TL;DR: This review is an attempt to correlate the structures of the 50S and 30S ribosomal subunits with biochemical and genetic data to identify the gaps and limits in current knowledge of the mechanisms involved in translation.
863 citations
TL;DR: The mechanisms conserved across the three kingdoms of life are discussed as well as the important divergences that have taken place in the pathway.
Abstract: ▪ Abstract Great advances have been made in the past three decades in understanding the molecular mechanics underlying protein synthesis in bacteria, but our understanding of the corresponding events in eukaryotic organisms is only beginning to catch up. In this review we describe the current state of our knowledge and ignorance of the molecular mechanics underlying eukaryotic translation. We discuss the mechanisms conserved across the three kingdoms of life as well as the important divergences that have taken place in the pathway.
588 citations
TL;DR: A comparison study of RNA binding in NMR-based and electrospray Ionization Mass Spectrometry-Based methods found that the former showed superior results while the latter showed better results in relation to the latter.
Abstract: 5.1. Binding Sites of the Aminoglycosides 1185 5.2. Importance of Electrostatic Interactions 1185 5.3. Nonionic Interactions 1185 5.4. Pseudo-Base Pair Interactions 1186 5.5. Water-Mediated Contacts 1186 5.6. Shape Complementarity and Conformational Adaptation 1186 6. Assays for Evaluating RNA Binding 1186 6.1. Methods Utilizing Fluorescently Labeled RNA 1186 6.2. NMR-Based Methods 1187 6.3. Electrospray Ionization Mass Spectrometry-Based Methods 1187
573 citations