scholarly journals Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 546 ◽  
Author(s):  
David Schwark ◽  
Margaret Schmitt ◽  
John Fisk
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Codon Reassignment ◽  
Methanocaldococcus Jannaschii ◽  
E Coli ◽  
Amber Stop Codon ◽  
Orthogonal Pair ◽  
Amber Codon ◽  
Quantitative Contribution ◽  
Stop Codon Reassignment

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor

2013 ◽  
Vol 91 (3) ◽  
pp. 155-164 ◽  
Author(s):  
Lijun Xu ◽  
Yanrong Hao ◽  
Cui Li ◽  
Quan Shen ◽  
Baofeng Chai ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Reassignment ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Terminal Domain ◽  
Class 1 ◽  
Stop Codon Recognition ◽  
Stop Codon Reassignment

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.

scholarly journals Sense codon reassignment enables viral resistance and encoded polymer synthesis

Science ◽  
2021 ◽  
Vol 372 (6546) ◽  
pp. 1057-1062
Author(s):  
Wesley E. Robertson ◽  
Louise F. H. Funke ◽  
Daniel de la Torre ◽  
Julius Fredens ◽  
Thomas S. Elliott ◽  
...  
Keyword(s):  
Stop Codon ◽  
Synonymous Codon ◽  
Viral Resistance ◽  
Release Factor ◽  
Codon Reassignment ◽  
Efficient Synthesis ◽  
Noncanonical Amino Acids ◽  
Laboratory Evolution ◽  
Transfer Rnas ◽  
Sense Codon

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)—making their cognate codons unreadable—might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

scholarly journals Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

scholarly journals ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Active Conformation ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

SummaryRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named ResQ. Genetic analysis shows that B. subtilis requires the function of either trans-translation or ResQ for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-EM characterization demonstrates that ResQ binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although ResQ is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

scholarly journals Connection between stop codon reassignment and frequent use of shifty stop frameshifting

RNA ◽  
2009 ◽  
Vol 15 (5) ◽  
pp. 889-897 ◽  
Author(s):  
H. Vallabhaneni ◽  
H. Fan-Minogue ◽  
D. M. Bedwell ◽  
P. J. Farabaugh
Keyword(s):  
Stop Codon ◽  
Codon Reassignment ◽  
Frequent Use ◽  
Stop Codon Reassignment

scholarly journals Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential

2021 ◽  
Vol 22 (2) ◽  
pp. 895
Author(s):  
David G. Schwark ◽  
Margaret A. Schmitt ◽  
John D. Fisk
Keyword(s):  
Genetic Code ◽  
Directed Evolution ◽  
Trna Synthetase ◽  
Methanosarcina Barkeri ◽  
Rapid Evaluation ◽  
Codon Reassignment ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Orthogonal Pair ◽  
Sense Codon

Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri orthogonal pair may infiltrate the E. coli genetic code.

scholarly journals Validation that human microbiome phages use alternative genetic coding with TAG stop read as Q

2022 ◽  
Author(s):  
Samantha Peters ◽  
Adair L Borges ◽  
Richard J Giannone ◽  
Michael Morowitz ◽  
Jill Banfield ◽  
...  
Keyword(s):  
Stop Codon ◽  
Human Microbiome ◽  
Structural Proteins ◽  
Codon Reassignment ◽  
Human Gut ◽  
Interaction Dynamics ◽  
Host Interaction ◽  
Genetic Codes ◽  
First Time ◽  
Stop Codon Reassignment

Metagenomic findings suggesting that bacteriophages (phages) can use genetic codes different from those of their host bacteria reveal a new dimension of phage-host interaction dynamics. Whereas reassignment of stop codons to code for amino acids has been predicted, there has been no proteomic validation of alternative coding in phages. In fact, one code where the stop codon TAG is reassigned to glutamine (code 15) has never been experimentally validated in any biological system. Here, we characterized stop codon reassignment in two crAss-like phages found in the human gut microbiome using LC-MS/MS-based metaproteomics. The proteome data from several phage structural proteins clearly demonstrates reassignment of the TAG stop codon to glutamine, establishing for the first time the expression of genetic code 15.

scholarly journals Stops making sense: translational trade-offs and stop codon reassignment

2011 ◽  
Vol 11 (1) ◽  
Author(s):  
Louise J Johnson ◽  
James A Cotton ◽  
Conrad P Lichtenstein ◽  
Greg S Elgar ◽  
Richard A Nichols ◽  
...  
Keyword(s):  
Stop Codon ◽  
Codon Reassignment ◽  
Making Sense ◽  
Trade Offs ◽  
Stop Codon Reassignment

Gene Overexpression as a Tool for Identifying New trans-Acting Factors Involved in Translation Termination in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 585-594
Author(s):  
Olivier Namy ◽  
Isabelle Hatin ◽  
Guillaume Stahl ◽  
Hongmei Liu ◽  
Stephanie Barnay ◽  
...  
Keyword(s):  
Protein Transport ◽  
Stop Codon ◽  
Translation Termination ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Release Factor ◽  
Lacz Reporter Gene ◽  
Pole Body ◽  
Secondary Phenotypes ◽  
Termination Process

Abstract In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.

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