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

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.

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

Science Advances ◽

10.1126/sciadv.aax8030 ◽

2019 ◽

Vol 5 (12) ◽

pp. eaax8030 ◽

Cited By ~ 6

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.

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Converting GTP hydrolysis into motion: versatile translational elongation factor G

Biological Chemistry ◽

10.1515/hsz-2019-0313 ◽

2019 ◽

Vol 401 (1) ◽

pp. 131-142 ◽

Cited By ~ 2

Author(s):

Marina V. Rodnina ◽

Frank Peske ◽

Bee-Zen Peng ◽

Riccardo Belardinelli ◽

Wolfgang Wintermeyer

Keyword(s):

Elongation Factor ◽

Ribosome Recycling Factor ◽

Canonical Function ◽

Elongation Factor G ◽

Translation Elongation ◽

Gtp Hydrolysis ◽

Reading Frame ◽

Gtpase Cycle ◽

A Site ◽

Factor G

Abstract Elongation factor G (EF-G) is a translational GTPase that acts at several stages of protein synthesis. Its canonical function is to catalyze tRNA movement during translation elongation, but it also acts at the last step of translation to promote ribosome recycling. Moreover, EF-G has additional functions, such as helping the ribosome to maintain the mRNA reading frame or to slide over non-coding stretches of the mRNA. EF-G has an unconventional GTPase cycle that couples the energy of GTP hydrolysis to movement. EF-G facilitates movement in the GDP-Pi form. To convert the energy of hydrolysis to movement, it requires various ligands in the A site, such as a tRNA in translocation, an mRNA secondary structure element in ribosome sliding, or ribosome recycling factor in post-termination complex disassembly. The ligand defines the direction and timing of EF-G-facilitated motion. In this review, we summarize recent advances in understanding the mechanism of EF-G action as a remarkable force-generating GTPase.

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EF-G–induced ribosome sliding along the noncoding mRNA

Science Advances ◽

10.1126/sciadv.aaw9049 ◽

2019 ◽

Vol 5 (6) ◽

pp. eaaw9049 ◽

Cited By ~ 5

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.

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Mechanism of tRNA-mediated +1 ribosomal frameshifting

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809319115 ◽

2018 ◽

Vol 115 (44) ◽

pp. 11226-11231 ◽

Cited By ~ 14

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.

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Insight into physicochemical properties of Nd2CuO4±δ and the A-site cation deficient Nd1.9CuO4±δ layered oxides

Functional Materials Letters ◽

10.1142/s1793604720510340 ◽

2020 ◽

Vol 13 (06) ◽

pp. 2051034

Author(s):

Bartłomiej Gȩdziorowski ◽

Anna Niemczyk ◽

Anna Olszewska ◽

Kacper Cichy ◽

Konrad Świerczek

Keyword(s):

Crystal Structure ◽

Electrical Conductivity ◽

Physicochemical Properties ◽

Oxygen Vacancies ◽

Numerical Density ◽

Interstitial Oxygen ◽

Layered Oxides ◽

Oxygen Defects ◽

A Site ◽

Insight Into

Nd2CuO[Formula: see text] is known to possess either oxygen vacancies or interstitial oxygen defects, depending on the synthesis route, as well as may exhibit the A-site deficiency. In this work, insight into physicochemical properties of Nd2CuO[Formula: see text] and Nd[Formula: see text]CuO[Formula: see text] layered oxides is given, focusing on the crystal structure, electrical conductivity, and oxygen permeation, as well as on numerical density functional theory (DFT) simulations concerning ionic defects formation and their possible movement in the crystal structure. The results indicate that in oxidizing conditions at low temperatures, interstitial oxygen defects are stable, but with the increase of temperature, the release of oxygen is observed, leading to formation of the oxygen vacancies. Both materials are stable at elevated temperatures in air and Ar. Larger oxygen nonstoichiometry and improved electrical conductivity at high temperatures for Nd[Formula: see text]CuO[Formula: see text] compound are accompanied by the recorded oxygen flux of ca. 0.2[Formula: see text]mL[Formula: see text]cm[Formula: see text][Formula: see text]min[Formula: see text] at 880∘C for 0.8[Formula: see text]mm thick ceramic membrane.

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The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

Journal of Biological Chemistry ◽

10.1074/jbc.m607058200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32303-32309 ◽

Cited By ~ 53

Author(s):

Taeko Komoda ◽

Neuza S. Sato ◽

Steven S. Phelps ◽

Naoki Namba ◽

Simpson Joseph ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Functional Role ◽

Elongation Factor ◽

Normal Growth ◽

Wild Type ◽

Reading Frame ◽

E Coli ◽

Translational Activity ◽

A Site

Helix 38 (H38) in 23 S rRNA, which is known as the “A-site finger (ASF),” is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

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Ribosome collisions alter frameshifting at translational reprogramming motifs in bacterial mRNAs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910613116 ◽

2019 ◽

Vol 116 (43) ◽

pp. 21769-21779 ◽

Cited By ~ 10

Author(s):

Angela M. Smith ◽

Michael S. Costello ◽

Andrew H. Kettring ◽

Robert J. Wingo ◽

Sean D. Moore

Keyword(s):

Ribosomal Protein ◽

Strong Dependence ◽

Elongation Factor ◽

Messenger Rnas ◽

Reading Frame ◽

Translational Frameshifting ◽

Naturally Occurring ◽

Mechanistic Insight ◽

Mrna Pool ◽

Insight Into

Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522932113 ◽

2016 ◽

Vol 113 (18) ◽

pp. 4994-4999 ◽

Cited By ~ 13

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.

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

Nature Communications ◽

10.1038/s41467-021-24911-1 ◽

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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