The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform

Wolfgang Wintermeyer - One of the best experts on this subject based on the ideXlab platform.

  • Synchronous tRNA movements durinG translocation on the ribosome are orchestrated by ElonGation Factor G and GTP hydrolysis.
    BioEssays : news and reviews in molecular cellular and developmental biology, 2014
    Co-Authors: Wolf Holtkamp, Wolfgang Wintermeyer, Marina V Rodnina
    Abstract:

    The translocation of tRNAs throuGh the ribosome proceeds throuGh numerous small steps in which tRNAs Gradually shift their positions on the small and larGe ribosomal subunits. The most urGent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase ElonGation Factor G (EF-G), promotes directed movement; and (iii) how the enerGy of GTP hydrolysis is coupled to movement. In the liGht of recent advances in biophysical and structural studies, we arGue that intermediate states of translocation are snapshots of dynamic fluctuations that Guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the enerGy reGimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms.

  • Dual use of GTP hydrolysis by ElonGation Factor G on the ribosome.
    Translation (Austin Tex.), 2013
    Co-Authors: Carlos E. Cunha, Frank Peske, Wolfgang Wintermeyer, Riccardo Belardinelli, Wolf Holtkamp, Marina V Rodnina
    Abstract:

    ElonGation Factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation durinG the ElonGation cycle of protein synthesis. The GTP-bound state of the Factor on the ribosome has been studied mainly with non-hydrolyzable analoGs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanGed affinity, allowinG us to study the function of the authentic GTP-bound form of EF-G in translocation. UtilizinG fluorescent reporter Groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suGGest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearranGements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the Factor. Thus, EF-G combines the enerGy reGime characteristic for motor proteins, acceleratinG movement by a conformational chanGe induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowinG the dissociation of the Factor.

  • Functions of ElonGation Factor G in translocation and ribosome recyclinG
    Ribosomes, 2011
    Co-Authors: Wolfgang Wintermeyer, Frank Peske, Andreas Savelsbergh, Vladimir I Katunin, Andrey L. Konevega, Yuri P. Semenkov, Niels Fischer, Holger Stark, Marina V Rodnina
    Abstract:

    AmonG the translation Factors that assist the ribosome in synthesizinG proteins, ElonGation Factor G (EF-G) is the only one that functions in two different phases of protein synthesis, i. e. in the translocation step of the ElonGation phase and in ribosome disassembly followinG termination. DurinG translocation two tRNAs move by larGe distances from one site of the ribosome to the next, adjacent site, with the coupled movement of mRNA by one codon. The process is promoted by EF-G and GTP hydrolysis to proceed at the velocity required for rapid protein synthesis in the cell. DurinG ribosome recyclinG the ribosomal post-termination complex is dissociated into subunits; the reaction is brouGht about by EF-G toGether with the ribosome recyclinG Factor (RRF) and requires GTP hydrolysis. Fundamental questions in understandinG EF-G function are: (i) How does EF-G accelerate the movement of tRNAs toGether with the mRNA on the ribosome; (ii) how does EF-G cooperate with RRF to dissociate the ribosomes; and (iii) how are GTP hydrolysis and Pi release coupled to forward movement and ribosome disassembly? The aim of this review is to summarize the recent insiGhts into the molecular mechanism of translocation and ribosome recyclinG and the role of EF-G in the two reactions. Detailed accounts focusinG on different aspects of translocation can also be found in several recent reviews (Shoji et al., 2009; Dunkle and Cate, 2010; Frank and Gonzalez, 2010).

  • distinct functions of ElonGation Factor G in ribosome recyclinG and translocation
    RNA, 2009
    Co-Authors: Andreas Savelsbergh, Marina V Rodnina, Wolfgang Wintermeyer
    Abstract:

    ElonGation Factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, toGether with ribosome recyclinG Factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is stronGly inhibited by vanadate, an analoG of inorGanic phosphate (Pi), indicatinG that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in sinGle-round translocation is not affected by vanadate, while the turnover reaction is stronGly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suGGestinG that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recyclinG. Our results indicate that conformational couplinG between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tiGht couplinG between the conformational chanGe of EF-G induced by Pi release and ribosome disassembly.

  • control of phosphate release from ElonGation Factor G by ribosomal protein l7 12
    The EMBO Journal, 2005
    Co-Authors: Andreas Savelsbergh, Wolfgang Wintermeyer, Dagmar Mohr, Ute Kothe, Marina V Rodnina
    Abstract:

    Ribosomal protein L7/12 is crucial for the function of ElonGation Factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. SinGle conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G·GTP for the ribosome, suGGestinG an involvement of these residues in EF-G bindinG. The mutations did not appreciably affect rapid sinGle-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorGanic phosphate (Pi) from ribosome-bound EF-G·GDP·Pi was stronGly inhibited and became rate-limitinG for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaininG the conformational couplinG between EF-G and the ribosome for translocation and for timinG the dissociation of the Factor from the ribosome.

Marina V Rodnina - One of the best experts on this subject based on the ideXlab platform.

  • Synchronous tRNA movements durinG translocation on the ribosome are orchestrated by ElonGation Factor G and GTP hydrolysis.
    BioEssays : news and reviews in molecular cellular and developmental biology, 2014
    Co-Authors: Wolf Holtkamp, Wolfgang Wintermeyer, Marina V Rodnina
    Abstract:

    The translocation of tRNAs throuGh the ribosome proceeds throuGh numerous small steps in which tRNAs Gradually shift their positions on the small and larGe ribosomal subunits. The most urGent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase ElonGation Factor G (EF-G), promotes directed movement; and (iii) how the enerGy of GTP hydrolysis is coupled to movement. In the liGht of recent advances in biophysical and structural studies, we arGue that intermediate states of translocation are snapshots of dynamic fluctuations that Guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the enerGy reGimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms.

  • Dual use of GTP hydrolysis by ElonGation Factor G on the ribosome.
    Translation (Austin Tex.), 2013
    Co-Authors: Carlos E. Cunha, Frank Peske, Wolfgang Wintermeyer, Riccardo Belardinelli, Wolf Holtkamp, Marina V Rodnina
    Abstract:

    ElonGation Factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation durinG the ElonGation cycle of protein synthesis. The GTP-bound state of the Factor on the ribosome has been studied mainly with non-hydrolyzable analoGs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanGed affinity, allowinG us to study the function of the authentic GTP-bound form of EF-G in translocation. UtilizinG fluorescent reporter Groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suGGest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearranGements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the Factor. Thus, EF-G combines the enerGy reGime characteristic for motor proteins, acceleratinG movement by a conformational chanGe induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowinG the dissociation of the Factor.

  • Functions of ElonGation Factor G in translocation and ribosome recyclinG
    Ribosomes, 2011
    Co-Authors: Wolfgang Wintermeyer, Frank Peske, Andreas Savelsbergh, Vladimir I Katunin, Andrey L. Konevega, Yuri P. Semenkov, Niels Fischer, Holger Stark, Marina V Rodnina
    Abstract:

    AmonG the translation Factors that assist the ribosome in synthesizinG proteins, ElonGation Factor G (EF-G) is the only one that functions in two different phases of protein synthesis, i. e. in the translocation step of the ElonGation phase and in ribosome disassembly followinG termination. DurinG translocation two tRNAs move by larGe distances from one site of the ribosome to the next, adjacent site, with the coupled movement of mRNA by one codon. The process is promoted by EF-G and GTP hydrolysis to proceed at the velocity required for rapid protein synthesis in the cell. DurinG ribosome recyclinG the ribosomal post-termination complex is dissociated into subunits; the reaction is brouGht about by EF-G toGether with the ribosome recyclinG Factor (RRF) and requires GTP hydrolysis. Fundamental questions in understandinG EF-G function are: (i) How does EF-G accelerate the movement of tRNAs toGether with the mRNA on the ribosome; (ii) how does EF-G cooperate with RRF to dissociate the ribosomes; and (iii) how are GTP hydrolysis and Pi release coupled to forward movement and ribosome disassembly? The aim of this review is to summarize the recent insiGhts into the molecular mechanism of translocation and ribosome recyclinG and the role of EF-G in the two reactions. Detailed accounts focusinG on different aspects of translocation can also be found in several recent reviews (Shoji et al., 2009; Dunkle and Cate, 2010; Frank and Gonzalez, 2010).

  • distinct functions of ElonGation Factor G in ribosome recyclinG and translocation
    RNA, 2009
    Co-Authors: Andreas Savelsbergh, Marina V Rodnina, Wolfgang Wintermeyer
    Abstract:

    ElonGation Factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, toGether with ribosome recyclinG Factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is stronGly inhibited by vanadate, an analoG of inorGanic phosphate (Pi), indicatinG that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in sinGle-round translocation is not affected by vanadate, while the turnover reaction is stronGly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suGGestinG that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recyclinG. Our results indicate that conformational couplinG between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tiGht couplinG between the conformational chanGe of EF-G induced by Pi release and ribosome disassembly.

  • control of phosphate release from ElonGation Factor G by ribosomal protein l7 12
    The EMBO Journal, 2005
    Co-Authors: Andreas Savelsbergh, Wolfgang Wintermeyer, Dagmar Mohr, Ute Kothe, Marina V Rodnina
    Abstract:

    Ribosomal protein L7/12 is crucial for the function of ElonGation Factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. SinGle conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G·GTP for the ribosome, suGGestinG an involvement of these residues in EF-G bindinG. The mutations did not appreciably affect rapid sinGle-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorGanic phosphate (Pi) from ribosome-bound EF-G·GDP·Pi was stronGly inhibited and became rate-limitinG for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaininG the conformational couplinG between EF-G and the ribosome for translocation and for timinG the dissociation of the Factor from the ribosome.

Andreas Savelsbergh - One of the best experts on this subject based on the ideXlab platform.

  • Functions of ElonGation Factor G in translocation and ribosome recyclinG
    Ribosomes, 2011
    Co-Authors: Wolfgang Wintermeyer, Frank Peske, Andreas Savelsbergh, Vladimir I Katunin, Andrey L. Konevega, Yuri P. Semenkov, Niels Fischer, Holger Stark, Marina V Rodnina
    Abstract:

    AmonG the translation Factors that assist the ribosome in synthesizinG proteins, ElonGation Factor G (EF-G) is the only one that functions in two different phases of protein synthesis, i. e. in the translocation step of the ElonGation phase and in ribosome disassembly followinG termination. DurinG translocation two tRNAs move by larGe distances from one site of the ribosome to the next, adjacent site, with the coupled movement of mRNA by one codon. The process is promoted by EF-G and GTP hydrolysis to proceed at the velocity required for rapid protein synthesis in the cell. DurinG ribosome recyclinG the ribosomal post-termination complex is dissociated into subunits; the reaction is brouGht about by EF-G toGether with the ribosome recyclinG Factor (RRF) and requires GTP hydrolysis. Fundamental questions in understandinG EF-G function are: (i) How does EF-G accelerate the movement of tRNAs toGether with the mRNA on the ribosome; (ii) how does EF-G cooperate with RRF to dissociate the ribosomes; and (iii) how are GTP hydrolysis and Pi release coupled to forward movement and ribosome disassembly? The aim of this review is to summarize the recent insiGhts into the molecular mechanism of translocation and ribosome recyclinG and the role of EF-G in the two reactions. Detailed accounts focusinG on different aspects of translocation can also be found in several recent reviews (Shoji et al., 2009; Dunkle and Cate, 2010; Frank and Gonzalez, 2010).

  • distinct functions of ElonGation Factor G in ribosome recyclinG and translocation
    RNA, 2009
    Co-Authors: Andreas Savelsbergh, Marina V Rodnina, Wolfgang Wintermeyer
    Abstract:

    ElonGation Factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, toGether with ribosome recyclinG Factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is stronGly inhibited by vanadate, an analoG of inorGanic phosphate (Pi), indicatinG that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in sinGle-round translocation is not affected by vanadate, while the turnover reaction is stronGly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suGGestinG that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recyclinG. Our results indicate that conformational couplinG between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tiGht couplinG between the conformational chanGe of EF-G induced by Pi release and ribosome disassembly.

  • control of phosphate release from ElonGation Factor G by ribosomal protein l7 12
    The EMBO Journal, 2005
    Co-Authors: Andreas Savelsbergh, Wolfgang Wintermeyer, Dagmar Mohr, Ute Kothe, Marina V Rodnina
    Abstract:

    Ribosomal protein L7/12 is crucial for the function of ElonGation Factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. SinGle conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G·GTP for the ribosome, suGGestinG an involvement of these residues in EF-G bindinG. The mutations did not appreciably affect rapid sinGle-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorGanic phosphate (Pi) from ribosome-bound EF-G·GDP·Pi was stronGly inhibited and became rate-limitinG for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaininG the conformational couplinG between EF-G and the ribosome for translocation and for timinG the dissociation of the Factor from the ribosome.

  • Control of phosphate release from ElonGation Factor G by ribosomal protein L7/12
    The EMBO journal, 2005
    Co-Authors: Andreas Savelsbergh, Wolfgang Wintermeyer, Dagmar Mohr, Ute Kothe, Marina V Rodnina
    Abstract:

    Ribosomal protein L7/12 is crucial for the function of ElonGation Factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. SinGle conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G·GTP for the ribosome, suGGestinG an involvement of these residues in EF-G bindinG. The mutations did not appreciably affect rapid sinGle-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorGanic phosphate (Pi) from ribosome-bound EF-G·GDP·Pi was stronGly inhibited and became rate-limitinG for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaininG the conformational couplinG between EF-G and the ribosome for translocation and for timinG the dissociation of the Factor from the ribosome.

  • conformational chanGes of the small ribosomal subunit durinG ElonGation Factor G dependent trna mrna translocation
    Journal of Molecular Biology, 2004
    Co-Authors: Frank Peske, Andreas Savelsbergh, Vladimir I Katunin, Marina V Rodnina, Wolfgang Wintermeyer
    Abstract:

    Translocation, a coordinated movement of two tRNAs toGether with mRNA on the ribosome, is catalyzed by ElonGation Factor G (EF-G). The reaction is accompanied by conformational rearranGements of the ribosome that are, as yet, not well characterized. Here, we analyze those rearranGements by restrictinG the conformational flexibility of the ribosome by antibiotics bindinG to specific sites of the ribosome. Paromomycin (Par), viomycin (Vio), spectinomycin (Spc), and hyGromycin B (HyGB) inhibited the tRNA-mRNA movement, while the other partial reactions of translocation, includinG the unlockinG rearranGement of the ribosome that precedes tRNA-mRNA movement, were not affected. The functional cycle of EF-G, i.e. bindinG of EF-G.GTP to the ribosome, GTP hydrolysis, Pi release, and dissociation of EF-G.GDP from the ribosome, was not affected either, indicatinG that EF-G turnover is not coupled directly to tRNA-mRNA movement. The inhibition of translocation by Par and Vio is attributed to the stabilization of tRNA bindinG in the A site, whereas Spc and HyGB had a direct inhibitory effect on tRNA-mRNA movement. Streptomycin (Str) had essentially no effect on translocation, althouGh it caused a larGe increase in tRNA affinity to the A site. These results suGGest that conformational chanGes in the vicinity of the decodinG reGion at the bindinG sites of Spc and HyGB are important for tRNA-mRNA movement, whereas Str seems to stabilize a conformation of the ribosome that is prone to rapid translocation, thereby compensatinG the effect on tRNA affinity.

Dagmar Mohr - One of the best experts on this subject based on the ideXlab platform.

  • control of phosphate release from ElonGation Factor G by ribosomal protein l7 12
    The EMBO Journal, 2005
    Co-Authors: Andreas Savelsbergh, Wolfgang Wintermeyer, Dagmar Mohr, Ute Kothe, Marina V Rodnina
    Abstract:

    Ribosomal protein L7/12 is crucial for the function of ElonGation Factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. SinGle conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G·GTP for the ribosome, suGGestinG an involvement of these residues in EF-G bindinG. The mutations did not appreciably affect rapid sinGle-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorGanic phosphate (Pi) from ribosome-bound EF-G·GDP·Pi was stronGly inhibited and became rate-limitinG for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaininG the conformational couplinG between EF-G and the ribosome for translocation and for timinG the dissociation of the Factor from the ribosome.

  • Control of phosphate release from ElonGation Factor G by ribosomal protein L7/12
    The EMBO journal, 2005
    Co-Authors: Andreas Savelsbergh, Wolfgang Wintermeyer, Dagmar Mohr, Ute Kothe, Marina V Rodnina
    Abstract:

    Ribosomal protein L7/12 is crucial for the function of ElonGation Factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. SinGle conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G·GTP for the ribosome, suGGestinG an involvement of these residues in EF-G bindinG. The mutations did not appreciably affect rapid sinGle-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorGanic phosphate (Pi) from ribosome-bound EF-G·GDP·Pi was stronGly inhibited and became rate-limitinG for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaininG the conformational couplinG between EF-G and the ribosome for translocation and for timinG the dissociation of the Factor from the ribosome.

  • an ElonGation Factor G induced ribosome rearranGement precedes trna mrna translocation
    Molecular Cell, 2003
    Co-Authors: Andreas Savelsbergh, Frank Peske, Vladimir I Katunin, Marina V Rodnina, Dagmar Mohr, Wolfgang Wintermeyer
    Abstract:

    The ElonGation cycle of protein synthesis is completed by translocation, a rearranGement durinG which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by ElonGation Factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suGGests that GTP hydrolysis drives a conformational rearranGement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the enerGy of GTP hydrolysis is utilized to promote the ribosome rearranGement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA.

  • ArGinines 29 and 59 of ElonGation Factor G are important for GTP hydrolysis or translocation on the ribosome.
    The EMBO journal, 2000
    Co-Authors: Dagmar Mohr, Wolfgang Wintermeyer, Marina V Rodnina
    Abstract:

    GTP hydrolysis by ElonGation Factor G (EF‐G) is essential for the translocation step in protein ElonGation. The low intrinsic GTPase activity of EF‐G is stronGly stimulated by the ribosome. Here we show that a conserved arGinine, R29, of Escherichia coli EF‐G is crucial for GTP hydrolysis on the ribosome, but not for GTP bindinG or ribosome interaction, suGGestinG that it may be directly involved in catalysis. Another conserved arGinine, R59, which is homoloGous to the catalytic arGinine of G α proteins, is not essential for GTP hydrolysis, but influences ribosome bindinG and translocation. These results indicate that EF‐G is similar to other GTPases in that an arGinine residue is required for GTP hydrolysis, althouGh the structural chanGes leadinG to GTPase activation are different.

Vladimir I Katunin - One of the best experts on this subject based on the ideXlab platform.

  • Functions of ElonGation Factor G in translocation and ribosome recyclinG
    Ribosomes, 2011
    Co-Authors: Wolfgang Wintermeyer, Frank Peske, Andreas Savelsbergh, Vladimir I Katunin, Andrey L. Konevega, Yuri P. Semenkov, Niels Fischer, Holger Stark, Marina V Rodnina
    Abstract:

    AmonG the translation Factors that assist the ribosome in synthesizinG proteins, ElonGation Factor G (EF-G) is the only one that functions in two different phases of protein synthesis, i. e. in the translocation step of the ElonGation phase and in ribosome disassembly followinG termination. DurinG translocation two tRNAs move by larGe distances from one site of the ribosome to the next, adjacent site, with the coupled movement of mRNA by one codon. The process is promoted by EF-G and GTP hydrolysis to proceed at the velocity required for rapid protein synthesis in the cell. DurinG ribosome recyclinG the ribosomal post-termination complex is dissociated into subunits; the reaction is brouGht about by EF-G toGether with the ribosome recyclinG Factor (RRF) and requires GTP hydrolysis. Fundamental questions in understandinG EF-G function are: (i) How does EF-G accelerate the movement of tRNAs toGether with the mRNA on the ribosome; (ii) how does EF-G cooperate with RRF to dissociate the ribosomes; and (iii) how are GTP hydrolysis and Pi release coupled to forward movement and ribosome disassembly? The aim of this review is to summarize the recent insiGhts into the molecular mechanism of translocation and ribosome recyclinG and the role of EF-G in the two reactions. Detailed accounts focusinG on different aspects of translocation can also be found in several recent reviews (Shoji et al., 2009; Dunkle and Cate, 2010; Frank and Gonzalez, 2010).

  • conformational chanGes of the small ribosomal subunit durinG ElonGation Factor G dependent trna mrna translocation
    Journal of Molecular Biology, 2004
    Co-Authors: Frank Peske, Andreas Savelsbergh, Vladimir I Katunin, Marina V Rodnina, Wolfgang Wintermeyer
    Abstract:

    Translocation, a coordinated movement of two tRNAs toGether with mRNA on the ribosome, is catalyzed by ElonGation Factor G (EF-G). The reaction is accompanied by conformational rearranGements of the ribosome that are, as yet, not well characterized. Here, we analyze those rearranGements by restrictinG the conformational flexibility of the ribosome by antibiotics bindinG to specific sites of the ribosome. Paromomycin (Par), viomycin (Vio), spectinomycin (Spc), and hyGromycin B (HyGB) inhibited the tRNA-mRNA movement, while the other partial reactions of translocation, includinG the unlockinG rearranGement of the ribosome that precedes tRNA-mRNA movement, were not affected. The functional cycle of EF-G, i.e. bindinG of EF-G.GTP to the ribosome, GTP hydrolysis, Pi release, and dissociation of EF-G.GDP from the ribosome, was not affected either, indicatinG that EF-G turnover is not coupled directly to tRNA-mRNA movement. The inhibition of translocation by Par and Vio is attributed to the stabilization of tRNA bindinG in the A site, whereas Spc and HyGB had a direct inhibitory effect on tRNA-mRNA movement. Streptomycin (Str) had essentially no effect on translocation, althouGh it caused a larGe increase in tRNA affinity to the A site. These results suGGest that conformational chanGes in the vicinity of the decodinG reGion at the bindinG sites of Spc and HyGB are important for tRNA-mRNA movement, whereas Str seems to stabilize a conformation of the ribosome that is prone to rapid translocation, thereby compensatinG the effect on tRNA affinity.

  • an ElonGation Factor G induced ribosome rearranGement precedes trna mrna translocation
    Molecular Cell, 2003
    Co-Authors: Andreas Savelsbergh, Frank Peske, Vladimir I Katunin, Marina V Rodnina, Dagmar Mohr, Wolfgang Wintermeyer
    Abstract:

    The ElonGation cycle of protein synthesis is completed by translocation, a rearranGement durinG which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by ElonGation Factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suGGests that GTP hydrolysis drives a conformational rearranGement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the enerGy of GTP hydrolysis is utilized to promote the ribosome rearranGement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA.

  • couplinG of Gtp hydrolysis by ElonGation Factor G to translocation and Factor recyclinG on the ribosome
    Biochemistry, 2002
    Co-Authors: Vladimir I Katunin, Andreas Savelsbergh, Marina V Rodnina, Wolfgang Wintermeyer
    Abstract:

    The translocation step of ElonGation entails the coordinated movement of tRNA and mRNA on the ribosome. Translocation is promoted by ElonGation Factor G (EF-G) and accompanied by GTP hydrolysis, which affects both translocation and turnover of EF-G. Both reactions are much slower (50−100-fold) when GTP is replaced with non-hydrolyzable GTP analoGues or GDP, indicatinG that the reaction rates are determined by conformational transitions induced by GTP hydrolysis. Compared to the rate of uncatalyzed, spontaneous translocation, ribosome bindinG of EF-G with any Guanine nucleotide reduces the free enerGy of activation by about 18 kJ/mol, whereas GTP hydrolysis contributes another 10 kJ/mol. The acceleration by GTP hydrolysis is due to larGe decrease in activation enthalpy by about 30 kJ/mol, compared to the reaction with GTP analoGues or GDP, whereas the activation entropy becomes unfavorable and is lowered by about 20 kJ/mol (37 °C). The data suGGest that GTP hydrolysis induces, by a conformational chanGe of...

  • Thiostrepton inhibits the turnover but not the GTPase of ElonGation Factor G on the ribosome
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Marina V Rodnina, Andreas Savelsbergh, Vladimir I Katunin, Yuri P. Semenkov, Natalia B. Matassova, Wolfgang Wintermeyer
    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.