Journal of Molecular Biology
Regular articleMissense translation errors in Saccharomyces cerevisiae1
Introduction
Translation of an mRNA does not always result in the synthesis of a full-length polypeptide chain of the correct length or with the correct amino acid sequence. At a low, but nevertheless detectable, level ribosomes can alter their reading frame (frameshift errors), prematurely terminate or fail to recognise the natural termination codon (termination errors), or simply insert the incorrect amino acid (missense error). Missense errors can occur either as a result of a non-cognate interaction between the tRNA anticodon and the mRNA codon, or due to mischarging of a tRNA with the incorrect amino acid by the amino-acyl tRNA synthetase (Parker, 1989; review). A further contribution to the cumulative gene decoding error frequency is made by inaccuracies in the process of transcription by the RNA polymerase, producing a flawed mRNA template, which the ribosomes then translate. Studies in Escherichia coli indicate that the average missense error frequency in the living prokaryotic cell is approximately 4×10−4 per codon translated, although estimates for specific decoding errors can range from 10−3 to 10−5 per codon translated (Parker, 1989). Frameshift and termination errors may occur up to an order of magnitude more frequently. In vivo errors in transcription in E. coli vary between 10−4 and 10−5 mistakes per base incorporated Rosenberger and Hilton 1983, Blank et al 1986.
Although over-large reductions in translational error frequencies can be accompanied by a kinetic penalty Gupta and Schessinger 1976, Piepersberg et al 1979, it is obviously essential for the cell to reduce these errors to a minimum, partly because the fitness of the cell machinery is reduced, but also because an enhanced translational error frequency can increase the spontaneous mutation rate as a result of the formation of mutator DNA polymerase molecules (Boe, 1992).
A number of attempts have been made to measure the frequencies of the various types of translational error in the bacterial cell, but in eukaryotes such assays have been restricted to measuring termination errors Martin et al 1989, Firoozan et al 1991 and frameshift errors (Stahl et al., 1995). The measurement of missense error frequencies in both prokaryotes and eukaryotes presents a much greater technical challenge. In vitro experiments can be conducted using cell-free translation extracts programmed with the artificial template poly(U) encoding poly(Phe) (codon UUU), and measuring the misincorporation of leucine (codon CUU). However, such experiments must be carried out at non-physiological Mg2+ concentrations (12 mM), making this type of assay less than ideal for accurate estimates of the endogenous missense error frequency in a living cell.
In vivo quantification of missense errors in E. coli has been approached in one of two ways. The first is to select a cellular protein that by chance is composed of only 19 of the possible 20 amino acids. Growth of the bacterial cell on defined medium where that amino acid missing from the protein is supplied in a radioactively labelled form allows the frequency of misincorporation to be measured accurately. For example, by purifying flagellin from E. coli a protein that contains no cysteine, a misreading frequency per codon of 1×10−4 was estimated, although the site of misincorporation could only be inferred (Edelmann & Gallant, 1977). This approach has been further refined to include a step in which proteolytic cleavage of the purified protein at a specific site is inhibited by amino acid misincorporation (Bouadloun et al., 1983). Alternatively, a protein containing such a radioactively labelled amino acid can be subjected to N-terminal sequencing (Parker et al., 1983).
The second way that missense errors can be quantified in vivo involves the use of inactive enzyme mutants that no longer exhibit catalytic activity with missense translation, resulting in the incorporation of the required residue and thus restoring enzyme activity. These types of assay fall into two categories, the “19-active” and “one-active” types (Cornut & Wilson, 1991). With the first type, any of 19 amino acids inserted at a given position result in significant enzyme activity, with the 20th eliminating activity. Such an assay will detect any possible mistranslation event and enable calculation of a global error frequency. Alternatively, one-active assays, in which only the wild-type residue exhibits activity and any of the other 19 amino acids do not, can only be used to quantify one specific type of missense event. However, these assays have the advantage that quite specific questions can be answered about the type of misreading and the mechanism of codon-anticodon interaction. In a one-active type of assay, the inactive mutant enzymes must exhibit a specific activity that is a fraction of the specific activity of the wild-type enzyme; that fraction must be at least an order of magnitude less than the error frequency being measured. If this condition is not met, the background activity of the mutant enzyme will mask the activity arising from those mistranslation events that result in the production of wild-type enzyme. A one-active assay based on a β-lactamase mutant, where the codon encoding the wild-type catalytic residue Ser68 residue was replaced by the glycine codon GGC, was used to demonstrate that first codon base mispairing occurred with a relatively high frequency in E. coli (1×10−3; Toth et al., 1988).
Estimates of missense translation error frequencies have focused almost exclusively on error events in E. coli, partly because of the availability of a number of mutants that either increase (e.g. ram mutants) or decrease (e.g.rpsL mutants) the levels of such errors (Gorini, 1974). Studies with the yeast Saccharomyces cerevisiae have also identified a number of mutants that have phenotypes consistent with an inability to control translational errors; suppression of nonsense mutations, hypersensitivity to translational-error-inducing antibiotics such as paromomycin, and error-prone ribosomes in cell-free mistranslation assays (Stansfield & Tuite, 1994; review). Although several of these mutants turned out to have mutations in genes encoding translation termination factors (Stansfield et al., 1995; review), several others had mutations in genes encoding ribosomal proteins, e.g. the SUP44 and SUP46 mutants Vincent and Liebman 1992, All-Robyn et al 1990.
In contrast to the situation in E. coli, very little is known of the corresponding missense error frequencies in any eukaryotic organism. The only in vivo measurement has been with rabbit reticulocytes, in which the misincorporation of valine at isoleucine codons was measured using tryptic fragments of purified haemoglobin; these experiments indicated the in vivo missense error frequency was approximately 3×10−4 (Loftfield & Vanderjagt, 1972). Corresponding in vitro experiments have measured the frequency of incorporation, into protamine, of radiolabelled amino acids not ordinarily encoded by the template, in a mouse liver in vitro translation extract. These experiments put the missense error frequency at between 6×10−4and 2×10−3 (Mori et al., 1985).
To date, no simple, quantitative, in vivo missense translation assay has been described for a genetically tractable eukaryotic organism such as yeast. We have therefore developed a novel and rapid, in vivo missense translation assay for the yeast S. cerevisiae, based on measurement of the enzymatic activity of mutants of type III chloramphenicol acetyl transferase (CATIII) that are known to be catalytically inactive.
Section snippets
An assay for measuring missense errors in yeast
A missense assay was developed based on the one-active strategy using catalytically inactive mutants of type III CAT (CATIII) (Lewendon et al., 1994). The CAT variant III from Shigella flexinirii was chosen over the more commonly used variant I from E. coli transposon Tn 9 because the three-dimensional X-ray crystal structure of CATIII has been solved and its mechanism of catalytic activity deduced (Leslie et al., 1988). Active site mutants of the essential catalytic residue His195 were chosen
Discussion
We have developed the first accurate and quantifiable in vivo assay for a specific misincorporation event in a eukaryotic cell, based on the misreading of a UAC (His) codon as a tyrosine codon in a bacterial CATIII gene expressed in yeast. The knowledge of the crystal structure of type III CAT, and subsequent deduction that its mechanism of catalysis is absolutely dependent upon the presence of a histidine residue at position 195, provided the basis of this one-active type of assay. The
Strains and growth media
Yeast strains (genotypes given in Table 1) were grown on either YEPD complete medium (2% (w/v) glucose, 2% (w/v) Bacto-peptone, 1% (w/v) yeast extract), or defined minimal (0.67% (w/v) Difco defined minimal without amino acids, 2% (w/v) glucose) supplemented with the appropriate amino acids and co-factors. Escherichia coli strain DH5α (F′/endA1 hsdR17 (rk− mk+) supE44 thi-1 recA1 gyrA (NaIR)relA1Δ (lacZYA-argF) U169 (φ80dlacΔ(lacZ) M15) was used for cloning experiments and cultured as described
Acknowledgements
The research described here was supported by a project grant from the BBSRC (Chemical & Pharmaceuticals Directorate) to M.F.T., and a Royal Society equipment grant to I.S. We thank Sue Liebman for the provision of the SUP44 and SUP46 strains, and Manuel Santos for reading the manuscript and giving helpful advice.
References (50)
- et al.
Mechanism of cycloheximide inhibition of protein synthesis in a cell-free system prepared from rat liver
J. Biol. Chem.
(1969) - et al.
Measurement of translational accuracy in vivomissense reporting using inactive enzyme mutants
Biochimie
(1991) - et al.
Mistranslation in E. coli
Cell
(1977) - et al.
Altered 40 S ribosomal subunits in omnipotent suppressors of yeast
J. Mol. Biol.
(1986) - et al.
Serine insertion caused by the ribosomal suppressor SUP46 in yeast
J. Mol. Biol.
(1981) - et al.
Codon usage and mistranslation - in vivo basal level misreading of the MS2 coat protein message
J. Biol. Chem.
(1983) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria
Methods Enzymol.
(1975)- et al.
The end in sightterminating translation in eukaryotes
Trends Biochem. Sci.
(1995) - et al.
Evidence for a unique first position codon-anticodon mismatch in vivo
J. Mol. Biol.
(1988) - et al.
The effects of paromomycin on the fidelity of translation in a yeast cell-free system
Biochim. Biophys. Acta
(1984)
Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader
J. Biol. Chem.
Cytosine 73 is a disciminator nucleotide in vivo for histidyl transfer RNA in E. coli
J. Biol. Chem.
Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein
Mol. Cell. Biol.
Electroporation in yeast
Methods Enzymol.
An RNA polymerase mutant with reduced accuracy of chain elongation
Biochemistry
Translational errors as the cause of mutations in Escherichia coli
Mol. Gen. Genet.
Codon-specific missense errors in vivo
EMBO J.
Patterns of genetic and phenotypic suppression of lys2 mutations in the yeast Saccharomyces cerevisiae
Genetics
Post-transcriptional nucleotide addition is responsible for the formation of the 5′ terminus of histidine tRNA
Proc. Natl Acad. Sci. USA
The translational signal database, TransTerm, is now a relational database
Nucl. Acids Res.
Quantification of readthrough of termination codons in yeast using a novel gene fusion assay
Yeast
Enzymatic aminoacylation of an eight base pair microhelix with histidine
Proc. Natl Acad. Sci. USA
Expression of genes transferred into monocot and dicot plants-cells by electroporation
Proc. Natl Acad. Sci. USA
Streptomycin and misreading of the genetic code
Coupling rates of transcription, translation and messenger ribonucleic acid degradation in streptomycin dependent mutants of E. coli
J. Bacteriol.
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Edited by K. Nagai
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Present address: I. Stansfield, Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.