scholarly journals EF-G–induced ribosome sliding along the noncoding mRNA

2019 ◽  
Vol 5 (6) ◽  
pp. eaaw9049 ◽  
Author(s):  
M. Klimova ◽  
T. Senyushkina ◽  
E. Samatova ◽  
B. Z. Peng ◽  
M. Pearson ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Elongation Factor ◽  
Transfer Rna ◽  
Noncoding Region ◽  
Forward Movement ◽  
Stem Loop ◽  
A Site ◽  
Hydrolysis Of ◽  
Reading Frames ◽  
Factor G

Translational bypassing is a recoding event during which ribosomes slide over a noncoding region of the messenger RNA (mRNA) to synthesize one protein from two discontinuous reading frames. Structures in the mRNA orchestrate forward movement of the ribosome, but what causes ribosomes to start sliding remains unclear. Here, we show that elongation factor G (EF-G) triggers ribosome take-off by a pseudotranslocation event using a small mRNA stem-loop as an A-site transfer RNA mimic and requires hydrolysis of about two molecules of guanosine 5′-triphosphate per nucleotide of the noncoding gap. Bypassing ribosomes adopt a hyper-rotated conformation, also observed with ribosomes stalled by the SecM sequence, suggesting common ribosome dynamics during translation stalling. Our results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to codon-wise ribosome movement during canonical translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs.

scholarly journals Active role of elongation factor G in maintaining the mRNA reading frame during translation

2019 ◽  
Vol 5 (12) ◽  
pp. eaax8030 ◽  
Author(s):  
Bee-Zen Peng ◽  
Lars V. Bock ◽  
Riccardo Belardinelli ◽  
Frank Peske ◽  
Helmut Grubmüller ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Active Role ◽  
Transfer Rna ◽  
Specific Interactions ◽  
Elongation Factor G ◽  
Reading Frame ◽  
A Site ◽  
Factor G ◽  
Domain 4

During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome’s control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation.

scholarly journals Mechanism of tRNA-mediated +1 ribosomal frameshifting

2018 ◽  
Vol 115 (44) ◽  
pp. 11226-11231 ◽  
Author(s):  
Samuel Hong ◽  
S. Sunita ◽  
Tatsuya Maehigashi ◽  
Eric D. Hoffer ◽  
Jack A. Dunkle ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Elongation Factor ◽  
Amino Acid Sequences ◽  
Structural Basis ◽  
Forward Movement ◽  
Stem Loop ◽  
Bacterial Ribosome ◽  
Conformational Rearrangements ◽  
A Site

Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA–mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

scholarly journals Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

2019 ◽  
Vol 116 (16) ◽  
pp. 7813-7818 ◽  
Author(s):  
Jie Zhou ◽  
Laura Lancaster ◽  
John Paul Donohue ◽  
Harry F. Noller
Keyword(s):  
Crystal Structure ◽  
Elongation Factor ◽  
Intermediate Complex ◽  
Reading Frame ◽  
Hybrid State ◽  
Head Domain ◽  
30S Subunit ◽  
A Site ◽  
Insight Into ◽  
Factor G

The elongation factor G (EF-G)–catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon–anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame.

scholarly journals Elongation factor G initiates translocation through a power stroke

2016 ◽  
Vol 113 (27) ◽  
pp. 7515-7520 ◽  
Author(s):  
Chunlai Chen ◽  
Xiaonan Cui ◽  
John F. Beausang ◽  
Haibo Zhang ◽  
Ian Farrell ◽  
...  
Keyword(s):  
Single Molecule ◽  
Messenger Rna ◽  
Elongation Factor ◽  
Power Stroke ◽  
Elongation Factor G ◽  
Gtp Hydrolysis ◽  
Guanosine Triphosphatase ◽  
Prokaryotic Protein ◽  
Domain Iii ◽  
Factor G

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

scholarly journals Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Jesper Pallesen ◽  
Yaser Hashem ◽  
Gürkan Korkmaz ◽  
Ravi Kiran Koripella ◽  
Chenhui Huang ◽  
...  
Keyword(s):  
G Protein ◽  
Messenger Rna ◽  
Stop Codon ◽  
Transfer Rna ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Rna Translation ◽  
Guanine Nucleotide Exchange ◽  
Messenger Rna Translation ◽  
A Site

Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.

scholarly journals Control of Ribosomal Subunit Rotation by Elongation Factor G

Science ◽  
2013 ◽  
Vol 340 (6140) ◽  
pp. 1235970 ◽  
Author(s):  
Arto Pulk ◽  
Jamie H. D. Cate
Keyword(s):  
Messenger Rna ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Elongation Factor G ◽  
Before And After ◽  
Switch Regions ◽  
Subunit Rotation ◽  
Guanosine Triphosphatase ◽  
Factor G

Protein synthesis by the ribosome requires the translocation of transfer RNAs and messenger RNA by one codon after each peptide bond is formed, a reaction that requires ribosomal subunit rotation and is catalyzed by the guanosine triphosphatase (GTPase) elongation factor G (EF-G). We determined 3 angstrom resolution x-ray crystal structures of EF-G complexed with a nonhydrolyzable guanosine 5′-triphosphate (GTP) analog and bound to the Escherichia coli ribosome in different states of ribosomal subunit rotation. The structures reveal that EF-G binding to the ribosome stabilizes switch regions in the GTPase active site, resulting in a compact EF-G conformation that favors an intermediate state of ribosomal subunit rotation. These structures suggest that EF-G controls the translocation reaction by cycles of conformational rigidity and relaxation before and after GTP hydrolysis.

scholarly journals A recent intermezzo at the Ribosome Club

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160185 ◽  
Author(s):  
Michael Y. Pavlov ◽  
Anders Liljas ◽  
Måns Ehrenberg
Keyword(s):  
Messenger Rna ◽  
Ternary Complex ◽  
Elongation Factor ◽  
Transfer Rna ◽  
Elongation Factor Tu ◽  
Transfer Rnas ◽  
30S Subunit ◽  
Translation Accuracy ◽  
Codon Selection ◽  
Two Sides

Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy ( d -values) and how the codon translation accuracy is reduced by the actions of Mg 2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals Elongation factor 4 remodels the A-site tRNA on the ribosome

2016 ◽  
Vol 113 (18) ◽  
pp. 4994-4999 ◽  
Author(s):  
Matthieu G. Gagnon ◽  
Jinzhong Lin ◽  
Thomas A. Steitz
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Thermus Thermophilus ◽  
Elongation Factor ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
70S Ribosome ◽  
Regulate Protein ◽  
A Site ◽  
Hydrolysis Of

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.

scholarly journals Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Demo ◽  
Howard B. Gamper ◽  
Anna B. Loveland ◽  
Isao Masuda ◽  
Christine E. Carbone ◽  
...  
Keyword(s):  
16S Rrna ◽  
Elongation Factor ◽  
Structural Basis ◽  
Elongation Factor G ◽  
Ribosomal Frameshifting ◽  
Elongation Cycle ◽  
70S Ribosome ◽  
Complex Features ◽  
A Site ◽  
Factor G

AbstractFrameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

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