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 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 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 Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

2020 ◽  
Author(s):  
Alexey Shuvalov ◽  
Ekaterina Shuvalova ◽  
Nikita Biziaev ◽  
Elizaveta Sokolova ◽  
Konstantin Evmenov ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Translation System ◽  
Release Factor ◽  
Viral Mrnas ◽  
Codon Recognition ◽  
Free Translation ◽  
Stop Codon Recognition ◽  
A Cell

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

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.

scholarly journals Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ

2013 ◽  
Vol 42 (5) ◽  
pp. 3152-3163 ◽  
Author(s):  
Hiroyuki Kogure ◽  
Yoshihiro Handa ◽  
Masahiro Nagata ◽  
Naoto Kanai ◽  
Peter Güntert ◽  
...  
Keyword(s):  
Solution Structure ◽  
Mutational Analysis ◽  
Single Amino Acid ◽  
Resonance Spectroscopy ◽  
Amino Acid Substitutions ◽  
Release Factor ◽  
E Coli ◽  
Ribosome Binding ◽  
A Site

Abstract The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.

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 60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5620 ◽  
Author(s):  
Sergey Levitskii ◽  
Ksenia Derbikova ◽  
Maria V. Baleva ◽  
Anton Kuzmenko ◽  
Andrey V. Golovin ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Sedimentation Coefficient ◽  
Initiation Factor ◽  
Dynamic State ◽  
Bacterial Cells ◽  
E Coli ◽  
Bacterial Ribosome

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of the ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for “60S state” formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.

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 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.

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