‘Stop’ in protein synthesis is modulated with exquisite subtlety by an extended RNA translation signal

2018 ◽  
Vol 46 (6) ◽  
pp. 1615-1625 ◽  
Author(s):  
Warren P. Tate ◽  
Andrew G. Cridge ◽  
Chris M. Brown
Keyword(s):  
Protein Synthesis ◽  
Conformational Changes ◽  
Stop Codon ◽  
Close Contact ◽  
Gc Content ◽  
Universal Genetic Code ◽  
Rna Translation ◽  
Stop Codons ◽  
Release Factors ◽  
Decoding Efficiency

Translational stop codons, UAA, UAG, and UGA, form an integral part of the universal genetic code. They are of significant interest today for their underlying fundamental role in terminating protein synthesis, but also for their potential utilisation for programmed alternative translation events. In diverse organisms, UAA has wide usage, but it is puzzling that the high fidelity UAG is selected against and yet UGA, vulnerable to suppression, is widely used, particularly in those archaeal and bacterial genomes with a high GC content. In canonical protein synthesis, stop codons are interpreted by protein release factors that structurally and functionally mimic decoding tRNAs and occupy the decoding site on the ribosome. The release factors make close contact with the decoding complex through multiple interactions. Correct interactions cause conformational changes resulting in new and enhanced contacts with the ribosome, particularly between specific bases in the mRNA and rRNA. The base following the stop codon (fourth or +4 base) may strongly influence decoding efficiency, facilitating alternative non-canonical events like frameshifting or selenocysteine incorporation. The fourth base is drawn into the decoding site with a compacted stop codon in the eukaryotic termination complex. Surprisingly, mRNA sequences upstream and downstream of this core tetranucleotide signal have a significant influence on the strength of the signal. Since nine bases downstream of the stop codon are within the mRNA channel, their interactions with rRNA, and r-proteins may affect efficiency. With this understanding, it is now possible to design stop signals of desired strength for specific applied purposes.

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.

scholarly journals Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity

2020 ◽  
Vol 117 (36) ◽  
pp. 22167-22172
Author(s):  
Hong Zhang ◽  
Zhihui Lyu ◽  
Yongqiang Fan ◽  
Christopher R. Evans ◽  
Karl W. Barber ◽  
...  
Keyword(s):  
Phenotypic Diversity ◽  
Stop Codon ◽  
Single Cells ◽  
Metabolic Stress ◽  
Aminoglycoside Antibiotic ◽  
Bacterial Cells ◽  
Excess Carbon ◽  
Stop Codons ◽  
Release Factors ◽  
Stop Codon Readthrough

Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.

scholarly journals Role of Premature Stop Codons in Bacterial Evolution

2008 ◽  
Vol 190 (20) ◽  
pp. 6718-6725 ◽  
Author(s):  
Tit-Yee Wong ◽  
Sanjit Fernandes ◽  
Naby Sankhon ◽  
Patrick P. Leong ◽  
Jimmy Kuo ◽  
...  
Keyword(s):  
Deinococcus Radiodurans ◽  
Stop Codon ◽  
Bacterial Species ◽  
Gc Content ◽  
Limited Range ◽  
Bacterial Evolution ◽  
Stop Codons ◽  
Patterns Of Use ◽  
Frequency Of Use ◽  
Gc Contents

ABSTRACT When the stop codons TGA, TAA, and TAG are found in the second and third reading frames of a protein-encoding gene, they are considered premature stop codons (PSC). Deinococcus radiodurans disproportionately favored TGA more than the other two triplets as a PSC. The TGA triplet was also found more often in noncoding regions and as a stop codon, though the bias was less pronounced. We investigated this phenomenon in 72 bacterial species with widely differing chromosomal GC contents. Although TGA and TAG were compositionally similar, we found a great variation in use of TGA but a very limited range of use of TAG. The frequency of use of TGA in the gene sequences generally increased with the GC content of the chromosome, while the frequency of use of TAG, like that of TAA, was inversely proportional to the GC content of the chromosome. The patterns of use of TAA, TGA and TAG as real stop codons were less biased and less influenced by the GC content of the chromosome. Bacteria with higher chromosomal GC contents often contained fewer PSC trimers in their genes. Phylogenetically related bacteria often exhibited similar PSC ratios. In addition, metabolically versatile bacteria have significantly fewer PSC trimers in their genes. The bias toward TGA but against TAG as a PSC could not be explained either by the preferential usage of specific codons or by the GC contents of individual chromosomes. We proposed that the quantity and the quality of the PSC in the genome might be important in bacterial evolution.

scholarly journals Distinct Paths To Stop Codon Reassignment by the Variant-Code Organisms Tetrahymena and Euplotes

2006 ◽  
Vol 26 (2) ◽  
pp. 438-447 ◽  
Author(s):  
Joe Salas-Marco ◽  
Hua Fan-Minogue ◽  
Adam K. Kallmeyer ◽  
Lawrence A. Klobutcher ◽  
Philip J. Farabaugh ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Yeast Cells ◽  
Universal Genetic Code ◽  
Stop Codons ◽  
Codon Recognition ◽  
Ciliate Species ◽  
Stop Codon Recognition ◽  
Euplotes Octocarinatus

ABSTRACT The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.

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 Stop codon recoding is widespread in diverse phage lineages and has the potential to regulate translation of late stage and lytic genes

2021 ◽  
Author(s):  
Adair L Borges ◽  
Yue Clare Lou ◽  
Rohan Sachdeva ◽  
Basem Al-Shayeb ◽  
Alexander L. Jaffe ◽  
...  
Keyword(s):  
Genetic Code ◽  
Late Stage ◽  
Stop Codon ◽  
Gc Content ◽  
Evolutionary Path ◽  
Stop Codons ◽  
Genetic Codes ◽  
Standard Code ◽  
And Control ◽  
Lytic Genes

The genetic code is a highly conserved feature of life. However, some alternative genetic codes use reassigned stop codons to code for amino acids. Here, we survey stop codon recoding across bacteriophages (phages) in human and animal gut microbiomes. We find that stop codon recoding has evolved in diverse clades of phages predicted to infect hosts that use the standard code. We provide evidence for an evolutionary path towards recoding involving reduction in the frequency of TGA and TAG stop codons due to low GC content, followed by acquisition of suppressor tRNAs and the emergence of recoded stop codons in structural and lysis genes. In analyses of two distinct lineages of recoded virulent phages, we find that lysis-related genes are uniquely biased towards use of recoded stop codons. This convergence supports the inference that stop codon recoding is a strategy to regulate the expression of late stage genes and control lysis timing. Interestingly, we identified prophages with recoded stop codons integrated into genomes of bacteria that use standard code, and hypothesize that recoding may control the lytic-lysogenic switch. Alternative coding has evolved many times, often in closely related lineages, indicating that genetic code is plastic in bacteriophages and adaptive recoding can occur over very short evolutionary timescales.

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 Translational readthrough goes unseen by natural selection

2019 ◽  
Author(s):  
April Snofrid Kleppe ◽  
Erich Bornberg-Bauer
Keyword(s):  
Protein Synthesis ◽  
Natural Selection ◽  
Selective Pressure ◽  
Evolutionary Rate ◽  
Stop Codon ◽  
Gc Content ◽  
Ribosome Profiling ◽  
Protein Isoforms ◽  
Common Error ◽  
Translational Readthrough

AbstractOccasionally during protein synthesis, the ribosome bypasses the stop codon and continues translation to the next stop codon in frame. This error is called translational readthrough (TR). Earlier research suggest that TR is a relatively common error, in several taxa, yet the evolutionary relevance of this translational error is still unclear. By analysing ribosome profiling data, we have conducted species comparisons between yeasts to infer conservation of TR between orthologs. Moreover, we infer the evolutionary rate of error prone and canonically translated proteins to deduct differential selective pressure. We find that about 40% of error prone proteins in Schizosaccharomyces pombe do not have any orthologs in Saccharomyces cerevisiae, but that 60% of error prone proteins in S. pombe are undergoing canonical translation in S. cerevisiae. Error prone proteins tend to have a higher GC-content in the 3’-UTR, unlike their canonically translated ortholog. We do not find the same trends for GC-content of the CDS. We discuss the role of 3’-UTR and GC-content regarding translational readthrough. Moreover, we find that there is neither selective pressure against or for TR. We suggest that TR is a near-neutral error that goes unseen by natural selection. We speculate that TR yield neutral protein isoforms that are not being purged. We suggest that isoforms, yielded by TR, increase proteomic diversity in the cell, which is readily available upon sudden environmental shifts and which therefore may become adaptive.Author SummaryThere is an evolutionary balance act between adaptation and selection against change. Any system needs to be able to adapt facing novel environmental conditions. Simultaneously, biological systems are under selection to maintain fitness and thus undergo selection against mutations. Phenotypic mutations - translational errors during protein synthesis - have been suggested to play a role in protein evolvability by enabling quick assessment of viable phenotypes and thus enable quick adaptation. Here we test this hypothesis, by inferring evolutionary rate of proteins prone to a specific case of phenotypic mutations: translational readthrough (TR). By making use of publicly available data of yeasts, we find that TR goes unseen by natural selection and appear as a neutral event. We suggest that TR goes unseen by selection and occurs as “permissive wallflowers”, which may become relevant and yield adaptive benefits. This work highlights that stochastic processes are not necessarily under stringent selection but may prevail. In conclusion, we suggest that TR is a neutral non-adaptive process that can yield adaptive benefits.

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.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

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