GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits

被引:75
作者
Holtkamp, Wolf [1 ]
Cunha, Carlos E. [1 ]
Peske, Frank [1 ]
Konevega, Andrey L. [1 ,2 ,3 ]
Wintermeyer, Wolfgang [1 ]
Rodnina, Marina V. [1 ]
机构
[1] Max Planck Inst Biophys Chem, Dept Phys Biochem, D-37077 Gottingen, Germany
[2] BP Konstantinov St Petersburg Nucl Phys Inst, Dept Mol & Radiat Biophys, Gatchina, Russia
[3] St Petersburg State Polytech Univ, Dept Biophys, St Petersburg, Russia
关键词
molecular machines; movement; protein synthesis; ribosome; translation; ELONGATION-FACTOR-G; PEPTIDYL-TRANSFER-RNA; HYBRID-STATE; PROTEIN-SYNTHESIS; 70S RIBOSOME; TRANSLATION ELONGATION; INTERSUBUNIT ROTATION; INTERMEDIATE STATES; ESCHERICHIA-COLI; SINGLE RIBOSOMES;
D O I
10.1002/embj.201387465
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G-GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.
引用
收藏
页码:1073 / 1085
页数:13
相关论文
共 83 条
[1]   Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome [J].
Agirrezabala, Xabier ;
Lei, Jianlin ;
Brunelle, Julie L. ;
Ortiz-Meoz, Rodrigo F. ;
Green, Rachel ;
Frank, Joachim .
MOLECULAR CELL, 2008, 32 (02) :190-197
[2]   Structural characterization of mRNA-tRNA translocation intermediates [J].
Agirrezabala, Xabier ;
Liao, Hstau Y. ;
Schreiner, Eduard ;
Fu, Jie ;
Ortiz-Meoz, Rodrigo F. ;
Schulten, Klaus ;
Green, Rachel ;
Frank, Joachim .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2012, 109 (16) :6094-6099
[3]   tRNA dynamics on the ribosome during translation [J].
Blanchard, SC ;
Kim, HD ;
Gonzalez, RL ;
Puglisi, JD ;
Chu, S .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (35) :12893-12898
[4]   Energy barriers and driving forces in tRNA translocation through the ribosome [J].
Bock, Lars V. ;
Blau, Christian ;
Schroeder, Gunnar F. ;
Davydov, Iakov I. ;
Fischer, Niels ;
Stark, Holger ;
Rodnina, Marina V. ;
Vaiana, Andrea C. ;
Grubmueller, Helmut .
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2013, 20 (12) :1390-1396
[5]   Structural basis for hygromycin B inhibition of protein biosynthesis [J].
Borovinskaya, Maria A. ;
Shoji, Shinichiro ;
Fredrick, Kurt ;
Cate, Jamie H. D. .
RNA, 2008, 14 (08) :1590-1599
[6]   A steric block in translation caused by the antibiotic spectinomycin [J].
Borovinskaya, Maria A. ;
Shoji, Shinichiro ;
Holton, James M. ;
Fredric, Kurt ;
Cate, Jamie H. D. .
ACS CHEMICAL BIOLOGY, 2007, 2 (08) :545-552
[7]   Truncated elongation factor G lacking the G domain promotes translocation of the 3' end but not of the anticodon domain in of peptidyl-tRNA [J].
Borowski, C ;
Rodnina, MV ;
Wintermeyer, W .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (09) :4202-4206
[8]   Structure of the ribosome with elongation factor G trapped in the pretranslocation state [J].
Brilot, Axel F. ;
Korostelev, Andrei A. ;
Ermolenko, Dmitri N. ;
Grigorieff, Nikolaus .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2013, 110 (52) :20994-20999
[9]   Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics [J].
Carter, AP ;
Clemons, WM ;
Brodersen, DE ;
Morgan-Warren, RJ ;
Wimberly, BT ;
Ramakrishnan, V .
NATURE, 2000, 407 (6802) :340-348
[10]   Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis [J].
Chen, Chunlai ;
Stevens, Benjamin ;
Kaur, Jaskiran ;
Smilansky, Zeev ;
Cooperman, Barry S. ;
Goldman, Yale E. .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2011, 108 (41) :16980-16985