Discrimination Between Defects in Elongation Fidelity and Termination Efficiency Provides Mechanistic Insights into Translational Readthrough

Salas-Marco, Joe; Bedwell, David M.

doi:10.1016/j.jmb.2005.03.025

Cited by 107 publications

(133 citation statements)

References 61 publications

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“…Using an extension of their system, we have attempted to analyze the full range of misreading errors by these tRNAs in S. cerevisiae. We confirm that the highest enzymatic activities reported at codon 245 by Salas-Marco and Bedwell (2005) reflect functional replacement rather than translational error but that half of the other mutants appear to have been incorrectly identified as error prone. For misreading by tRNA Lys UUU at codon 529 we show that, as in E. coli, error frequency is not solely determined by codon d anticodon pairing but rather is influenced by competition for decoding between cognate and near-cognate tRNAs.…”

Section: à4supporting

confidence: 69%

“…Again, most of these studies measured one or a few distinct errors at different codons, involving different mismatches, and at all three positions of the codondanticodon complex. For example, Salas-Marco and Bedwell (2005) (Salas-Marco and Bedwell 2005). The highest activities they recorded they ascribed to replacement of the wild-type histidine by the encoded mutant amino acid.…”

Section: à4mentioning

confidence: 99%

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A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

Kramer¹,

Vallabhaneni²,

Mayer³

et al. 2010

RNA

115

122

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The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 3 10 À5 to a high of 6.9 3 10 À4 per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.

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Section: à4supporting

confidence: 69%

Section: à4mentioning

confidence: 99%

A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

Kramer¹,

Vallabhaneni²,

Mayer³

et al. 2010

RNA

115

122

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“…It was reported earlier using a dual-luciferase reporter system that in uL11-depleted cells the frequency of reading-through events is elevated. 23 This phenomenon can also be explained by the loss of cognate vs. near-cognate discrimination (as discussed above). In eukaryotic cells, recognition of the STOP codon is subjected to the decoding process analogously to TC recognition, with the eRF1:eRF3:GTP complex (termination ternary complex tTC) as a main player, which structurally mimics TC, where eRF3 works as trGTPase analogously to EF-Tu.…”

Section: Ul11 Involvement In Ribosomal Speed and Accuracymentioning

confidence: 96%

“…22 The in vitro translation elongation rate measured by the poly-phenylalanine synthesis is decreased, whereas the decoding error rate, shown as a higher frequency of misincorporation events measured in vivo by a specific dual-luciferase system, is increased upon uL11 deletion. 23 Although the involvement of eukaryotic uL11 in ribosomal action was documented, the exact impact on the translational apparatus and metabolic fitness is still beyond full understanding. Moreover, apart from the direct role in the translational cycle, many eukaryotic ribosomal proteins were found to play an important role in ribosome biogenesis, a multistep process involving myriads of auxiliary trans-acting factors helping the ribosome to gain its final shape.…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of the uL11 protein impact on translational machinery

Wawiórka¹,

Molestak²,

Szajwaj³

et al. 2016

Cell Cycle

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The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of 'ribosomal life'. uL11 is involved in 'birth' (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the 'active life' of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase 'productive state' stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.

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“…Paromomycin is a 4,5 disubstituted aminoglycoside that increases misincorporation and suppresses nonsense mutations in yeast (Palmer et al 1979;Singh et al 1979;Chernoff et al 1994;Salas-Marco and Bedwell 2005). Due to the dramatic differences in the MIC of paromomycin for strains carrying these different rRNA mutations, we first grew strains expressing wild-type, G1645A, or G1645C 18S rRNAs in the presence of 25 mg/mL paromomycin.…”

Section: Decoding Site Mutations Alter Aminoglycoside Resistance In Ymentioning

confidence: 99%

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

Fan-Minogue¹,

Bedwell²

2007

RNA

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Recent studies of prokaryotic ribosomes have dramatically increased our knowledge of ribosomal RNA (rRNA) structure, functional centers, and their interactions with antibiotics. However, much less is known about how rRNA function differs between prokaryotic and eukaryotic ribosomes. The core decoding sites are identical in yeast and human 18S rRNAs, suggesting that insights obtained in studies with yeast rRNA mutants can provide information about ribosome function in both species. In this study, we examined the importance of key nucleotides of the 18S rRNA decoding site on ribosome function and aminoglycoside susceptibility in Saccharomyces cerevisiae cells expressing homogeneous populations of mutant ribosomes. We found that residues G577, A1755, and A1756 (corresponding to Escherichia coli residues G530, A1492, and A1493, respectively) are essential for cell viability. We also found that residue G1645 (A1408 in E. coli ) and A1754 (G1491 in E. coli ) both make significant and distinct contributions to aminoglycoside resistance. Furthermore, we found that mutations at these residues do not alter the basal level of translational accuracy, but influence both paromomycin-induced misreading of sense codons and readthrough of stop codons. This study represents the most comprehensive mutational analysis of the eukaryotic decoding site to date, and suggests that many fundamental features of decoding site function are conserved between prokaryotes and eukaryotes.

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Discrimination Between Defects in Elongation Fidelity and Termination Efficiency Provides Mechanistic Insights into Translational Readthrough

Cited by 107 publications

References 61 publications

A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

Functional analysis of the uL11 protein impact on translational machinery

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

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