Translational termination efficiency in both bacteria and mammals is regulated by the base following the stop codon

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1095-1103 ◽  
Author(s):  
Warren P. Tate ◽  
Elizabeth S. Poole ◽  
Julie A. Horsfield ◽  
Sally A. Mannering ◽  
Chris M. Brown ◽  
...  
Keyword(s):  
Stop Codon ◽  
Stop Signal ◽  
Physical Contact ◽  
Release Factor ◽  
Wide Range ◽  
Highly Expressed Genes ◽  
Peptidyltransferase Center ◽  
A Site ◽  
Translational Termination

The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl–tRNA hydrolysis activity and enhanced codon–ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where −1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol −1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.Key words: translational stop signal, decoding, release factor, frame-shifting.

scholarly journals Eukaryotic Release Factor 1 Phosphorylation by CK2 Protein Kinase Is Dynamic but Has Little Effect on the Efficiency of Translation Termination in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (8) ◽  
pp. 1378-1387 ◽  
Author(s):  
Adam K. Kallmeyer ◽  
Kim M. Keeling ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Number Of Factors ◽  
Stop Codon Recognition

ABSTRACT Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Δ mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

scholarly journals RiboA: a web application to identify ribosome A-site locations in ribosome profiling data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Danying Shao ◽  
Nabeel Ahmed ◽  
Nishant Soni ◽  
Edward P. O’Brien
Keyword(s):  
Integer Programming ◽  
Web Application ◽  
Stop Codon ◽  
Ribosome Profiling ◽  
Programming Method ◽  
Analysis Tool ◽  
Site Location ◽  
Link Type ◽  
Wide Range ◽  
A Site

Abstract Background Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations. Results We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms. Conclusions The Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/. The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.

scholarly journals Ribosome Recycling Factor and Release Factor 3 Action Promotes TnaC-Peptidyl-tRNA Dropoff and Relieves Ribosome Stalling during Tryptophan Induction of tna Operon Expression in Escherichia coli

2007 ◽  
Vol 189 (8) ◽  
pp. 3147-3155 ◽  
Author(s):  
Ming Gong ◽  
Luis R. Cruz-Vera ◽  
Charles Yanofsky
Keyword(s):  
Escherichia Coli ◽  
Growth Inhibition ◽  
Stop Codon ◽  
Elongation Factor ◽  
Initiation Factor ◽  
Release Factor ◽  
Ribosome Recycling Factor ◽  
Ribosome Stalling ◽  
Operon Expression

ABSTRACT Upon tryptophan induction of tna operon expression in Escherichia coli, the leader peptidyl-tRNA, TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document} , resists cleavage, resulting in ribosome stalling at the tnaC stop codon. This stalled ribosome blocks Rho factor binding and action, preventing transcription termination in the tna operon's leader region. Plasmid-mediated overexpression of tnaC was previously shown to inhibit cell growth by reducing uncharged \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document}  availability. Which factors relieve ribosome stalling, facilitate TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document}  cleavage, and relieve growth inhibition were addressed in the current study. In strains containing the chromosomal tna operon and lacking a tnaC plasmid, the overproduction of ribosome recycling factor (RRF) and release factor 3 (RF3) reduced tna operon expression. Their overproduction in vivo also increased the rate of cleavage of TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document} , relieving the growth inhibition associated with plasmid-mediated tnaC overexpression. The overproduction of elongation factor G or initiation factor 3 did not have comparable effects, and tmRNA was incapable of attacking TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document}  in stalled ribosome complexes. The stability of TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document}  was increased appreciably in strains deficient in RRF and RF3 or deficient in peptidyl-tRNA hydrolase. These findings reveal the existence of a natural mechanism whereby an amino acid, tryptophan, binds to ribosomes that have just completed the synthesis of TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document} . Bound tryptophan inhibits RF2-mediated cleavage of TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document} , resulting in the stalling of the ribosome translating tnaC mRNA. This stalling results in increased transcription of the structural genes of the tna operon. RRF and RF3 then bind to this stalled ribosome complex and slowly release TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document} . This release allows ribosome recycling and permits the cleavage of TnaC- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{2}^{Pro}\) \end{document}  by peptidyl-tRNA hydrolase.

scholarly journals Mechanism of ribosome rescue by ArfA and RF2

2016 ◽  
Author(s):  
Gabriel Demo ◽  
Egor Svidritskiy ◽  
Rohini Madireddy ◽  
Ruben Diaz-Avalos ◽  
Timothy Grant ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Stop Codon ◽  
Release Factor ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
C Terminus ◽  
Peptide Release ◽  
N Terminus ◽  
70S Ribosome ◽  
A Site

AbstractArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA∙RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation, hitherto unobserved in 70S termination complexes. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA “senses” the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

The Stop Signal Controls the Efficiency of Release Factor-Mediated Translational Termination

1996 ◽  
pp. 157-182 ◽  
Author(s):  
Warren P. Tate ◽  
Mark E. Dalphin ◽  
Herman J. Pel ◽  
Sally A. Mannering
Keyword(s):  
Stop Signal ◽  
Release Factor ◽  
Translational Termination

scholarly journals Characterization of late gene expression factors lef-9 and lef-8 from Bombyx mori nucleopolyhedrovirus

2002 ◽  
Vol 83 (8) ◽  
pp. 2015-2023 ◽  
Author(s):  
Asha Acharya ◽  
Karumathil P. Gopinathan
Keyword(s):  
Gene Expression ◽  
Bombyx Mori ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Early Transcription ◽  
A Site ◽  
Translational Termination

Late gene expression factors, LEF-4, LEF-8, LEF-9 and P47 constitute the primary components of the Autographa californica multinucleocapsid polyhedrovirus (AcMNPV)-encoded RNA polymerase, which initiates transcription from late and very late promoters. Here, characterization of lef-9 and lef-8, which encode their corresponding counterparts, from Bombyx mori NPV is reported. Transcription of lef-9 initiated at two independent sites: from a GCACT sequence located at −38 nt and a CTCTT sequence located at −50 nt, with respect to the +1 ATG of the open reading frame. The 3′ end of the transcript was mapped to a site 17 nt downstream of a canonical polyadenylation signal located 7 nt downstream of the first of the two tandem translational termination codons. Maximum synthesis of LEF-9 was seen from 36 h post-infection (p.i.). The transcription of lef-8 initiated early in infection from a GTGCAAT sequence that differed in the corresponding region from its AcMNPV counterpart (GCGCAGT), with consequent elimination of the consensus early transcription start site motif (underlined). Peak levels of lef-8 transcripts were attained by 24 h p.i. Immunocopurification analyses suggested that there was an association between LEF-8 and LEF-9 in vivo.

scholarly journals Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release

2018 ◽  
Vol 115 (3) ◽  
pp. E382-E389 ◽  
Author(s):  
Thomas Philipp Hoernes ◽  
Nina Clementi ◽  
Michael Andreas Juen ◽  
Xinying Shi ◽  
Klaus Faserl ◽  
...  
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Stop Codons ◽  
Codon Recognition ◽  
Chemically Modified ◽  
Peptide Release ◽  
Stop Codon Recognition ◽  
A Site ◽  
Chemical Groups ◽  
Insight Into

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

scholarly journals Comprehensive Analysis of Stop Codon Usage in Bacteria and Its Correlation with Release Factor Abundance

2014 ◽  
Vol 289 (44) ◽  
pp. 30334-30342 ◽  
Author(s):  
Gürkan Korkmaz ◽  
Mikael Holm ◽  
Tobias Wiens ◽  
Suparna Sanyal
Keyword(s):  
Codon Usage ◽  
Stop Codon ◽  
Gc Content ◽  
Comprehensive Analysis ◽  
Expression Level ◽  
Release Factor ◽  
Relative Level ◽  
Stop Codons ◽  
Release Factors ◽  
Highly Expressed Genes

We present a comprehensive analysis of stop codon usage in bacteria by analyzing over eight million coding sequences of 4684 bacterial sequences. Using a newly developed program called “stop codon counter,” the frequencies of the three classical stop codons TAA, TAG, and TGA were analyzed, and a publicly available stop codon database was built. Our analysis shows that with increasing genomic GC content the frequency of the TAA codon decreases and that of the TGA codon increases in a reciprocal manner. Interestingly, the release factor 1-specific codon TAG maintains a more or less uniform frequency (∼20%) irrespective of the GC content. The low abundance of TAG is also valid with respect to expression level of the genes ending with different stop codons. In contrast, the highly expressed genes predominantly end with TAA, ensuring termination with either of the two release factors. Using three model bacteria with different stop codon usage (Escherichia coli, Mycobacterium smegmatis, and Bacillus subtilis), we show that the frequency of TAG and TGA codons correlates well with the relative steady state amount of mRNA and protein for release factors RF1 and RF2 during exponential growth. Furthermore, using available microarray data for gene expression, we show that in both fast growing and contrasting biofilm formation conditions, the relative level of RF1 is nicely correlated with the expression level of the genes ending with TAG.

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