Missense translation errors in Saccharomyces cerevisiae

Stansfield, Ian; Jones, Kerrie M.; Herbert, Pamela; Lewendon, Ann; Shaw, William V.; Tuite, Mick F.

doi:10.1006/jmbi.1998.1976

Cited by 76 publications

(76 citation statements)

References 47 publications

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“…As a positive indicator of the efficacy of the assay presented here, we induced translational misreading of a reporter transcript in yeast using an antibiotic known to alter stop codon read-through. In prior work, paromomycin increased translation errors by ϳ20-fold at 200 M in yeast (20). Our ability to detect an effect of paromomycin in this reporter assay indicates that we can observe rate changes of this size.…”

Section: Discussionsupporting

confidence: 52%

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

Shaw

Bonawitz

Reines

2002

Journal of Biological Chemistry

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Eukaryotic RNA polymerase II and Escherichia coli RNA polymerase possess an intrinsic ribonuclease activity that is stimulated by the polymerase-binding proteins SII and GreB, respectively. This factor-activated hydrolysis of nascent RNA has been postulated to be involved in transcription elongation as well as removal of incorrect bases misincorporated into RNA. Little is known about the frequency of misincorporation by RNA polymerases in vivo or about the mechanisms involved in improving RNA polymerase accuracy. Here we have developed a luciferase reporter system in an effort to assay for base misincorporation in living Saccharomyces cerevisiae. The assay employs a luciferase open reading frame that contains a premature stop codon. The inactive truncated enzyme would become active if misincorporation by RNA polymerase II took place at the stop triplet. Yeast lacking SII did not display a significant change in reporter activity when compared with wild-type cells. We estimate that under our assay conditions, mRNAs with a misincorporation at the test site could not exceed 1 transcript per 500 cells. The reporter assay was very effective in detecting the previously described process of nonsense suppression (translational read-through) by ribosomes, making it difficult to determine an absolute level of basal (SII-independent) misincorporation by RNA polymerase II. Although these data cannot exclude the possibility that SII is involved in proofreading, they make it unlikely that such a contribution is physiologically significant, especially relative to the high frequency of translational errors.Many DNA polymerases contain a nuclease activity that allows them to excise, from newly replicated DNA, bases that are misincorporated with respect to Watson-Crick base pair rules. With the recognition that many, if not all, multisubunit DNA-dependent RNA polymerases contain a nuclease activity that operates on nascent RNA came the suggestion that RNA polymerases proofread RNA misincorporation events using this activity (1-3). For bacterial RNA polymerase, this nuclease activity is stimulated by small RNA polymerase-binding proteins called GreA and GreB (4). A similar activity in eukaryotic RNA polymerases is stimulated by a small RNA polymerase II-binding protein called SII (also known as TFIIS) that has been found in all eukaryotes so far investigated. The greA and greB genes are not essential for Escherichia coli; nor is the SII-encoding gene essential for Saccharomyces cerevisiae (5, 6). In vitro, these proteins can reactivate RNA polymerase enzymes that lapse into an elongation-incompetent form (7). They do so by activating cleavage of the nascent RNA chain by RNA polymerase. The vast majority of nascent RNA is assembled according to strict Watson-Crick base pairing rules. Hence, these proteins have been considered transcription elongation factors. In vivo evidence consistent with this role has been described (8 -14).In vitro, misincorporation of nucleotides into RNA by RNA polymerase can be detected by experimental manip...

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Section: Discussionsupporting

confidence: 52%

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

Shaw

Bonawitz

Reines

2002

Journal of Biological Chemistry

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“…Even fewer estimates of misreading rates are available for eukaryotic organisms. The few studies that have been done in vivo in the yeast Saccharomyces cerevisiae reported error frequencies ranging from z10 À3 to 10 À5 per codon (Stansfield et al 1998;Rakwalska and Rospert 2004;SalasMarco and Bedwell 2005;Plant et al 2007). 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.…”

Section: à4mentioning

confidence: 99%

“…The effect on misreading is much more specific than in E. coli, where paromomycin induced errors at all error-prone codons and some that did not show error in its absence (Kramer and Farabaugh 2007). Stansfield et al (1998) measured the effect of paromomycin on misreading by tRNA His GUG at a near cognate Tyr codon, UAC. That study found a 50-fold increase in error, a much greater effect than we have found.…”

Section: à5mentioning

confidence: 99%

“…This conclusion is not consistent with the fact that elimination of the AGG-decoding cognate tRNA causes a large increase in error; that result is more consistent with errors at AGG caused by misreading by tRNA Lys . Although misreading frequency has been reported to be as high as 1.5 3 10 À3 in yeast (Rakwalska and Rospert 2004), most studies have estimated the frequency to be more on the order of 10 À5 -10 À4 (Stansfield et al 1998;Salas-Marco and Bedwell 2005;Plant et al 2007). Our findings that tRNA Lys1 misreads near-cognate codons at a frequency of 8 3 10 À5 -7 3 10 À4 agree.…”

Section: à5mentioning

confidence: 99%

“…The range of misreading frequencies of the equivalent E. coli tRNA species is 2 3 10 À4 -4 3 10 À3 (Kramer and Farabaugh 2007), almost an order of magnitude higher. Stansfield et al (1998) suggested that E. coli might be intrinsically more error-prone than yeast. They reported a misreading of the first codon position in yeast that was almost two orders of magnitude lower than several reports of first position errors in E. coli, although the errors compared were different.…”

Section: à5mentioning

confidence: 99%

See 2 more Smart Citations

A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

Kramer¹,

Vallabhaneni²,

Mayer³

et al. 2010

RNA

111

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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Nonsense Mutations and Suppression

Herrington

2013

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 one of the chain‐termination codons, 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) such 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, in tRNA, or in translation factors can increase readthrough by altering the initial selection steps, proofreading or quality control in decoding of 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 changes associated with decoding and thus enhance or reduce readthrough of nonsense codons.

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Missense translation errors in Saccharomyces cerevisiae

Cited by 76 publications

References 47 publications

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

Nonsense Mutations and Suppression

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