Distinct Roles for Release Factor 1 and Release Factor 2 in Translational Quality Control

Πετρόπουλος, Αλέξανδρος Δ.; McDonald, Megan; Green, Rachel; Zaher, Hani

doi:10.1074/jbc.m114.564989

Cited by 34 publications

(32 citation statements)

References 36 publications

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“…To measure the kinetics of peptide release by ArfA and RF2, we used a peptide release assay similar to the one described by Green and colleagues (Zaher and Green 2009;Petropoulos et al 2014) with modifications (see Materials and Methods). This assay follows the rate of release of f-[…”

Section: Resultsmentioning

confidence: 99%

“…35 S]-Met-tRNA fMet , and ArfAΔ12C were formed using methods similar to one that has been used for the formation of canonical termination complexes (Zaher and Green 2009;Petropoulos et al 2014). Different concentrations of ArfAΔ12C were tested to ensure that a stable association of ArfAΔ12C to the ribosome was achieved ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Ribosomal complexes with P-site fMet-tRNA fMet were formed by incubating ribosome (2 µM), mRNA (6 µM), IF1, IF2, IF3 (3 µM each), GTP (2 mM), and fMet-tRNA fMet (3 µM) in buffer B at 37°C for 30 min (Zaher and Green 2009;Petropoulos et al 2014). The reactions were then pelleted through 0.5 mL of 1 M sucrose cushion in buffer B at 176,800g in a Type 45 Ti rotor for 24 h (Zaher and Green 2009).…”

Section: Release Complex Formationmentioning

confidence: 99%

“…All E. coli ribosomes and proteins except IF2 were stored in buffer B (50 mM Tris, pH 7.4, 70 mM NH 4 Cl, 30 mM KCl, 10 mM MgCl 2 , An induced-fit mechanism for nonstop termination www.rnajournal.org 57 and 5 mM β-mercaptoethanol) (Zaher and Green 2009;Petropoulos et al 2014). E. coli IF2 was stored in buffer C (50 mM HEPES, pH 7.6, 1 M NH 4 Cl, 10 mM MgCl 2 , and 5 mM β-mercaptoethanol).…”

Section: Ribosome Preparation Translation Initiation and Release Famentioning

confidence: 99%

“…In any case, decoding by RF2 appears to be more complicated than that of RF1 (Petropoulos et al 2014). Furthermore, a so-called "negativeto-positive charge-flip" mutation E167K in RF2 was reported to trigger the peptide release not only at the three conserved stop codons, but also at the sense codons such as UGG, UAC, and UUA (Ito et al 1998).…”

Section: Introductionmentioning

confidence: 99%

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Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Zeng

Jin

2015

RNA

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Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2 m ), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k cat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the k cat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Release Complex Formationmentioning

confidence: 99%

Section: Ribosome Preparation Translation Initiation and Release Famentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Zeng

Jin

2015

RNA

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Nonsense Mutations and Suppression

Herrington

2018

Encyclopedia of Life Sciences

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A nonsense mutation occurs when a sense codon, one that codes for an amino acid, is changed to a chain‐termination codon, UAG, UAA or UGA. A nonsense suppressor can result from a second mutation affecting the translational apparatus. This mutation enables the cell to insert an amino acid in response to the nonsense codon, resulting in a wild‐type or near wild‐type phenotype. Some suppressor mutations change the anticodon of a transfer ribonucleic acid (tRNA) so that it can pair with the nonsense codon. Other suppressors increase the readthrough of the nonsense mutation. Readthrough occurs at low levels but changes in the ribosome, tRNA or in translation factors can increase readthrough by altering the initial selection steps, proofreading or quality control in decoding the messenger RNA. Manipulation of the accuracy of translation holds promise as a method for the treatment of genetic diseases, many of which result from nonsense mutations. Key Concepts Suppression results when one mutation counteracts the effect of another mutation to give a wild‐type phenotype. The nonsense codons UAG, UAA and UGA do not code for amino acids, but signal the end of the protein‐coding sequence in the mRNA. Nonsense suppression competes with chain termination. Errors occur during translation and include reading a nonsense codon as sense as well as misreading and frameshifting. Decoding during elongation involves conformational changes in the ribosome, tRNA and EF‐Tu. Changes in the ribosome or other components of the translational apparatus can modify decoding and thus enhance or reduce readthrough of nonsense codons.

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Non‐canonical amino acid bioincorporation into antimicrobial peptides and its challenges

Enninful,

Kuppusamy,

Tiburu

et al. 2024

Journal of Peptide Science

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The rise of antimicrobial resistance and multi‐drug resistant pathogens has necessitated explorations for novel antibiotic agents as the discovery of conventional antibiotics is becoming economically less viable and technically more challenging for biopharma. Antimicrobial peptides (AMPs) have emerged as a promising alternative because of their particular mode of action, broad spectrum and difficulty that microbes have in becoming resistant to them. The AMPs bacitracin, gramicidin, polymyxins and daptomycin are currently used clinically. However, their susceptibility to proteolytic degradation, toxicity profile, and complexities in large‐scale manufacture have hindered their development. To improve their proteolytic stability, methods such as integrating non‐canonical amino acids (ncAAs) into their peptide sequence have been adopted, which also improves their potency and spectrum of action. The benefits of ncAA incorporation have been made possible by solid‐phase peptide synthesis. However, this method is not always suitable for commercial production of AMPs because of poor yield, scale‐up difficulties, and its non‐‘green’ nature. Bioincorporation of ncAA as a method of integration is an emerging field geared towards tackling the challenges of solid‐phase synthesis as a green, cheaper, and scalable alternative for commercialisation of AMPs. This review focusses on the bioincorporation of ncAAs; some challenges associated with the methods are outlined, and notes are given on how to overcome these challenges. The review focusses particularly on addressing two key challenges: AMP cytotoxicity towards microbial cell factories and the uptake of ncAAs that are unfavourable to them. Overcoming these challenges will draw us closer to a greater yield and an environmentally friendly and sustainable approach to make AMPs more druggable.

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Distinct Roles for Release Factor 1 and Release Factor 2 in Translational Quality Control

Cited by 34 publications

References 36 publications

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Nonsense Mutations and Suppression

Non‐canonical amino acid bioincorporation into antimicrobial peptides and its challenges

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