Decoding the Decoding Region: Analysis of Eukaryotic Release Factor (eRF1) Stop Codon-Binding Residues

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.

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

Nature Communications ◽

10.1038/s41467-019-13408-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

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.

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

Genetics ◽

10.1093/genetics/161.2.585 ◽

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

Eukaryotic Cell ◽

10.1128/ec.00073-06 ◽

2006 ◽

Vol 5 (8) ◽

pp. 1378-1387 ◽

Cited By ~ 12

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.

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Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals

Nucleic Acids Research ◽

10.1093/nar/26.4.954 ◽

1998 ◽

Vol 26 (4) ◽

pp. 954-960 ◽

Cited By ~ 49

Author(s):

E. Poole

Keyword(s):

Escherichia Coli ◽

Stop Codon ◽

Release Factor ◽

Translational Termination ◽

Decoding Efficiency

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

10.1101/732420 ◽

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.

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Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

eLife ◽

10.7554/elife.62655 ◽

2020 ◽

Vol 9 ◽

Author(s):

Kyle Mangano ◽

Tanja Florin ◽

Xinhao Shao ◽

Dorota Klepacki ◽

Irina Chelysheva ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Stop Codon ◽

Translation Termination ◽

Bacterial Cells ◽

Release Factor ◽

Translation Arrest ◽

Genome Wide Analysis ◽

Genome Wide ◽

Exit Tunnel ◽

Unique Mechanism

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

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

Genes ◽

10.3390/genes9110546 ◽

2018 ◽

Vol 9 (11) ◽

pp. 546 ◽

Cited By ~ 9

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.

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