scholarly journals Decision letter: Dynamics of ribosomes and release factors during translation termination in E. coli

2018 ◽  
Keyword(s):  
Translation Termination ◽  
E Coli ◽  
Release Factors

scholarly journals Author response: Dynamics of ribosomes and release factors during translation termination in E. coli

2018 ◽  
Author(s):  
Sarah Adio ◽  
Heena Sharma ◽  
Tamara Senyushkina ◽  
Prajwal Karki ◽  
Cristina Maracci ◽  
...  
Keyword(s):  
Translation Termination ◽  
Author Response ◽  
Response Dynamics ◽  
E Coli ◽  
Release Factors

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.

scholarly journals Dynamics of ribosomes and release factors during translation termination in E. coli

2018 ◽  
Author(s):  
Sarah Adio ◽  
Heena Sharma ◽  
Tamara Senyushkina ◽  
Prajwal Karki ◽  
Cristina Maracci ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Termination ◽  
Bound State ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Release Factors ◽  
Single Molecule Fret ◽  
Peptide Release ◽  
Two Factors ◽  
Stochastic Assembly

AbstractRelease factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

scholarly journals Methylation of E. coli Class I Release Factors Increases the Accuracy of Translation Termination in vitro

2018 ◽  
Author(s):  
Gürkan Korkmaz
Keyword(s):  
Translation Termination ◽  
Class I ◽  
Release Factor ◽  
E Coli ◽  
Release Factors ◽  
Peptide Release ◽  
Ribosomal Protein Synthesis ◽  
Uncompetitive Inhibitor ◽  
Menten Constant

ABSTRACTRibosomal protein synthesis (translation) is a highly accurate process. Translation termination, in particular, must be accurate to prevent truncated proteins. How this accuracy is achieved is not fully understood in all its details. Using an E. coli in vitro system, I explore novel mechanisms that contribute to the high accuracy of translation termination. By comparing the Michaelis-Menten parameters of methylated and non-methylated release factors on cognate and non-cognate codons. Post-translational methylation of a strictly conserved GGQ motif in class I release factors increases the accuracy of termination by up to 5-fold. This happens by increasing both the maximum rate of peptide release (kcat) and Michaelis-Menten constant (KM). Further, I demonstrate here that a non-methylated release factor acts like an uncompetitive inhibitor of enzyme reactions. Overall, this study shows that the methylation of class I release factors is a novel mechanism contributing to highly accurate translation termination.AbbreviationsRFrelease factorRCrelease complex

scholarly journals Extensive Ribosome and RF2 Rearrangements during Translation Termination

2019 ◽  
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

AbstractProtein 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.

scholarly journals Dynamics of ribosomes and release factors during translation termination in E. coli

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sarah Adio ◽  
Heena Sharma ◽  
Tamara Senyushkina ◽  
Prajwal Karki ◽  
Cristina Maracci ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Termination ◽  
Bound State ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Release Factors ◽  
Single Molecule Fret ◽  
Biochemical Assays ◽  
Peptide Release ◽  
Two Factors

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

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 An rRNA Fragment and Its Antisense Can Alter Decoding of Genetic Information

1998 ◽  
Vol 180 (10) ◽  
pp. 2744-2748 ◽  
Author(s):  
Alexey L. Arkov ◽  
Alexander Mankin ◽  
Emanuel J. Murgola
Keyword(s):  
Antisense Rna ◽  
Genetic Information ◽  
Translation Termination ◽  
23S Rrna ◽  
Release Factor ◽  
Expression Library ◽  
Nonsense Mutations ◽  
New Approach ◽  
E Coli

ABSTRACT rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in E. coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.

Termination of translation in eukaryotes

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1079-1086 ◽  
Author(s):  
Lev L. Kisselev ◽  
Lyudmila Yu. Frolova
Keyword(s):  
Polypeptide Chain ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
High Rate ◽  
Vitro Assay ◽  
Binding Motifs ◽  
Stop Codons ◽  
Release Factors ◽  
Terminal Domain ◽  
Gtp Binding

Termination of translation is governed in ribosomes by polypeptide chain release factors (pRF and eRF in prokaryotes and eukaryotes, respectively). In prokaryotes, three pRF have been identified and sequenced, while in eukaryotes, only a single eRF has been identified to date. Recently, we have characterized a highly conserved protein family called eRF1. At least, human and Xenopus laevis proteins from this family are active as eRFs in the in vitro assay with any of the three stop codons. No structural similarity has been revealed between any of the three pRFs and eRF1 family. Furthermore, GTP-binding motifs have not been revealed, although translation termination in eukaryotes is a GTP-dependent process. We have demonstrated that in eukaryotes a second eRF exists in addition to eRF1, called eRF3. The eRF3 family has two features in common: presence of GTP-binding motifs and high conservation of the C-terminal domain structure. The C-terminal domain of the X. laevis eRF3 has no RF activity although it stimulates the eRF1 activity considerably at low concentration of the stop codons, conferring GTP dependence to the termination reaction. Without eRF3, the eRF1 activity is entirely GTP independent. Some features of X. laevis eRF3 (C-terminal domain) resemble those of pRF3. The newly identified eRF1 and eRF3 are structurally conserved and distinct from the respective pRF1/2 and pRF3 proteins, pointing to the possibility of different evolution of translation termination machinery in prokaryotes and eukaryotes. Bipartition of the translation termination apparatus probably provides high rate and accuracy of translation termination.Key words: higher eukaryotic polypeptide chain release factors, translation termination, protein biosynthesis.

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