scholarly journals Bacterial ribosome conformational dynamics during translation termination and rescue

2017 ◽  
Author(s):  
◽  
Widler Casy
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Conformational Dynamics ◽  
Class I ◽  
Translation Inhibition ◽  
Release Factors ◽  
Bacterial Ribosome ◽  
A Site ◽  
Hydrolysis Of

Hydrolysis of polypeptide from the ribosome is a critical step that must occur prior to the ribosome recycling phase of translation. Inability of cells to do so can result in translation inhibition and eventually leading to cell death. In bacteria, class one release factors bind to the ribosome to catalyze the release of the mature polypeptide during translation termination. However, in the event of ribosome stalling as a result of mRNA truncation, ribosome rescue factors bind to the ribosome to catalyze the release of the growing polypeptide from the stalled complex. This rescue process is then followed by ribosome recycling. Here we employ smFRET to study the effects of the class I release factors, RF1 and RF2, and an alternative release factor known as YaeJ on the conformational dynamics of the ribosome following hydrolysis of peptidyl tRNAs. Further, we investigated the role of A-site mRNA on the global conformation of the ribosome. Our results demonstrate that upon binding to their cognate stop codon, the class I release factors stabilize ribosome complexes in the non-rotated state. Similarly, binding of YaeJ to complexes that are assembled on truncated mRNAs resulted in ribosomes that occupy primarily the non-rotated state. We also observe that absence of mRNA in the A-site induces a hyper-rotated conformation between the two subunits. Together, these findings further characterize the interactions between these different ligands and the bacterial ribosome. In addition, these results suggest that stabilization of the ribosome in the non-rotated state is critical for priming the ribosome for the recycling phase of translation.

scholarly journals The Origin and Evolution of Release Factors: Implications for Translation Termination, Ribosome Rescue and Quality Control Pathways

2019 ◽  
Author(s):  
A.Maxwell Burroughs ◽  
L Aravind
Keyword(s):  
Quality Control ◽  
Translation Termination ◽  
Last Universal Common Ancestor ◽  
Origin And Evolution ◽  
Release Factors ◽  
Universal Common Ancestor ◽  
Translation Apparatus ◽  
Ph Domains ◽  
Evolutionary Trajectories ◽  
Hydrolysis Of

The evolution of release factors catalyzing the hydrolysis of the final peptidyl-tRNA bond and the release of the polypeptide from the ribosome has been a longstanding paradox. While the components of the translation apparatus are generally well-conserved across extant life, structurally-unrelated release factor peptidyl hydrolases (RF-PHs) emerged in the stems of the bacterial and archaeo-eukaryotic lineages. We analyze the diversification of RF-PH domains within the broader evolutionary framework of the translation apparatus. Thus, we reconstruct the possible state of translation termination in the Last Universal Common Ancestor with possible tRNA-like terminators. Further, evolutionary trajectories of the several auxiliary release factors in ribosome quality control (RQC) and rescue pathways point to multiple independent solutions to this problem and frequent transfers between superkingdoms including the recently-characterized ArfT, which is more widely-distributed across life than previously appreciated. The eukaryotic RQC system was pieced together from components with disparate provenance, which include the long-sought Vms1/ANKZF1 RF-PH of bacterial origin. We also uncover an under-appreciated evolutionary driver of innovation in rescue pathways: effectors deployed in biological conflicts that target the ribosome. At least three rescue pathways (centered on the prfH/RFH, baeRF-1, and C12orf65 RF-PH domains), were likely innovated in response to such conflicts.

scholarly journals The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy

2006 ◽  
Vol 281 (7) ◽  
pp. 4548-4556 ◽  
Author(s):  
Lamine Bouakaz ◽  
Elli Bouakaz ◽  
Emanuel J. Murgola ◽  
Måns Ehrenberg ◽  
Suparna Sanyal
Keyword(s):  
Ribosomal Protein ◽  
Translation Termination ◽  
Class I ◽  
Release Factor ◽  
Translational Accuracy ◽  
Ribosomal Protein L11

scholarly journals Repurposing tRNAs for nonsense suppression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Suki Albers ◽  
Bertrand Beckert ◽  
Marco C. Matthies ◽  
Chandra Sekhar Mandava ◽  
Raphael Schuster ◽  
...  
Keyword(s):  
De Novo ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Nonsense Suppression ◽  
Potent Effect ◽  
Systematic Analysis ◽  
Stop Codons ◽  
Release Factors ◽  
A Site ◽  
Sense Codon

AbstractThree stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

scholarly journals Ribosome dynamics during decoding

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160182 ◽  
Author(s):  
Marina V. Rodnina ◽  
Niels Fischer ◽  
Cristina Maracci ◽  
Holger Stark
Keyword(s):  
Translation Termination ◽  
Elongation Factor ◽  
Translation Elongation ◽  
Gtp Hydrolysis ◽  
Codon Recognition ◽  
Special Cases ◽  
Multistep Process ◽  
Gtpase Activation ◽  
Hydrolysis Of

Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNA Sec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals Inhibition of translation termination by small molecules targeting ribosomal release factors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xueliang Ge ◽  
Ana Oliveira ◽  
Karin Hjort ◽  
Terese Bergfors ◽  
Hugo Gutiérrez-de-Terán ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Small Molecules ◽  
Translation Termination ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Release Factors ◽  
Bacterial Ribosome ◽  
Peptide Release ◽  
Bacterial Protein Synthesis ◽  
Important Drug

Abstract The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

Regulation of translation termination: conserved structural motifs in bacterial and eukaryotic polypeptide release factors

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1113-1122 ◽  
Author(s):  
Yoshikazu Nakamura ◽  
Koichi Ito ◽  
Kiyoyuki Matsumura ◽  
Yoichi Kawazu ◽  
Kanae Ebihara
Keyword(s):  
Functional Organization ◽  
Stop Codon ◽  
Translation Termination ◽  
Domain Model ◽  
Rna Recognition ◽  
Release Factor ◽  
Structural Motifs ◽  
Release Factors ◽  
Codon Recognition ◽  
Stop Codon Recognition

Translation termination requires codon-dependent polypeptide release factors. The mechanism of stop codon recognition by release factors is unknown and holds considerable interest since it entails protein–RNA recognition rather than the well-understood mRNA–tRNA interaction in codon–anticodon pairing. Bacteria have two codon-specific release factors and our picture of prokaryotic translation is changing because a third factor, which stimulates the other two, has now been found. Moreover, a highly conserved eukaryotic protein family possessing properties of polypeptide release factor has now been sought. This review summarizes our current understanding of the structural and functional organization of release factors as well as our recent findings of highly conserved structural motifs in bacterial and eukaryotic polypeptide release factors.Key words: translation termination, stop codon recognition, peptide chain release factors, seven-domain model.

The C domain of translation termination factor eRF1 is close to the stop codon in the A site of the 80S ribosome

2007 ◽  
Vol 41 (5) ◽  
pp. 781-789 ◽  
Author(s):  
K. N. Bulygin ◽  
E. A. Popugaeva ◽  
M. N. Repkova ◽  
M. I. Meschaninova ◽  
A. G. Ven’yaminova ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
80S Ribosome ◽  
Termination Factor ◽  
A Site ◽  
Translation Termination Factor

scholarly journals Methylation of Bacterial Release Factors RF1 and RF2 Is Required for Normal Translation Termination in Vivo

2007 ◽  
Vol 282 (49) ◽  
pp. 35638-35645 ◽  
Author(s):  
Liliana Mora ◽  
Valérie Heurgué-Hamard ◽  
Miklos de Zamaroczy ◽  
Stephanie Kervestin ◽  
Richard H. Buckingham
Keyword(s):  
Translation Termination ◽  
Carbon Sources ◽  
Ribosomal Subunit ◽  
Optimal Growth ◽  
Release Factors ◽  
Rich Media ◽  
Termination Activity ◽  
Hydrolysis Of

Bacterial release factors RF1 and RF2 are methylated on the Gln residue of a universally conserved tripeptide motif GGQ, which interacts with the peptidyl transferase center of the large ribosomal subunit, triggering hydrolysis of the ester bond in peptidyl-tRNA and releasing the newly synthesized polypeptide from the ribosome. In vitro experiments have shown that the activity of RF2 is stimulated by Gln methylation. The viability of Escherichia coli K12 strains depends on the integrity of the release factor methyltransferase PrmC, because K12 strains are partially deficient in RF2 activity due to the presence of a Thr residue at position 246 instead of Ala. Here, we study in vivo RF1 and RF2 activity at termination codons in competition with programmed frameshifting and the effect of the Ala-246 → Thr mutation. PrmC inactivation reduces the specific termination activity of RF1 and RF2(Ala-246) by ∼3- to 4-fold. The mutation Ala-246 → Thr in RF2 reduces the termination activity in cells ∼5-fold. After correction for the decrease in level of RF2 due to the autocontrol of RF2 synthesis, the mutation Ala-246 → Thr reduced RF2 termination activity by ∼10-fold at UGA codons and UAA codons. PrmC inactivation had no effect on cell growth in rich media but reduced growth considerably on poor carbon sources. This suggests that the expression of some genes needed for optimal growth under such conditions can become growth limiting as a result of inefficient translation termination.

scholarly journals Extensive ribosome and RF2 rearrangements during translation termination

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Egor Svidritskiy ◽  
Gabriel Demo ◽  
Anna B Loveland ◽  
Chen Xu ◽  
Andrei A Korostelev
Keyword(s):  
Protein Synthesis ◽  
Stop Codon ◽  
Translation Termination ◽  
Single Sample ◽  
Peptidyl Transferase ◽  
E Coli ◽  
Peptidyl Transferase Center ◽  
Release Factors ◽  
Subunit Rotation ◽  
Intersubunit Rotation

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long β-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

The Role of Conformational Dynamics and Allostery in Modulating Protein Evolution

2020 ◽  
Vol 49 (1) ◽  
pp. 267-288 ◽  
Author(s):  
Paul Campitelli ◽  
Tushar Modi ◽  
Sudhir Kumar ◽  
S. Banu Ozkan
Keyword(s):  
Protein Evolution ◽  
Environmental Changes ◽  
Conformational Dynamics ◽  
Amino Acid Level ◽  
Single Amino Acid ◽  
Dynamic Coupling ◽  
Coupling Index ◽  
A Site ◽  
Flexibility Index

Advances in sequencing techniques and statistical methods have made it possible not only to predict sequences of ancestral proteins but also to identify thousands of mutations in the human exome, some of which are disease associated. These developments have motivated numerous theories and raised many questions regarding the fundamental principles behind protein evolution, which have been traditionally investigated horizontally using the tip of the phylogenetic tree through comparative studies of extant proteins within a family. In this article, we review a vertical comparison of the modern and resurrected ancestral proteins. We focus mainly on the dynamical properties responsible for a protein's ability to adapt new functions in response to environmental changes. Using the Dynamic Flexibility Index and the Dynamic Coupling Index to quantify the relative flexibility and dynamic coupling at a site-specific, single-amino-acid level, we provide evidence that the migration of hinges, which are often functionally critical rigid sites, is a mechanism through which proteins can rapidly evolve. Additionally, we show that disease-associated mutations in proteins often result in flexibility changes even at positions distal from mutational sites, particularly in the modulation of active site dynamics.

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