A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae

Abstract

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 x 10(-5) to a high of 6.9 x 10(-4) 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.

FIGURE 1.

Map of the dual luciferase reporter plasmid pDB688. The plasmid pDB688 includes a single open reading frame encoding a fusion of the Renilla (Rluc), and firefly (Fluc) luciferase is shown flanked by the promoter of the constitutively expressed PGK gene and the poly(A) addition site of the CYH2 genes, both from S. cerevisiae. The location of a segment of the 2 μ circle that confers autonomous replication in S. cerevisiae and the URA3⁺ selectable gene are also indicated.

FIGURE 2.

Near- and noncognate mutations for lysine-529 (K529). The two Lys codons are shown in italics in this standard genetic code table. Near-cognate codons, differing at one codon position, are shown in reverse on black. Noncognate codons differ from Lys codons in more than one codon position; noncognate codons synonymous with tested near-cognate codons are shown in black on gray. The control UUU codon is shown in boldface.

FIGURE 3.

Variation in Fluc activity of K529 mutants. The graph shows the Fluc expression of the indicated constructs expressed as a fraction of the expression of wild-type Fluc. The activities for mutants that according to ANOVA are not significantly different from each other or the negative control are shown in black and the two activities that are significantly higher (P <0.05) are shown in white; the negative control UUU mutant is shown in gray. Indicated below each codon is the amino acid it encodes. Error bars are standard error of the mean.

FIGURE 4.

High residual activity of two K529 mutants results from near-cognate decoding. A comparison of the Fluc activity relative to wild type for synonymous near- and noncognate codon mutants are shown for the three nonsense mutants, UAA, UAG, and UGA, and six Arg mutants, AGA, AGG, CGU, CGC, CGA, and CGG. The data are represented as in Figure 3.

FIGURE 5.

Deletion of the single gene encoding the AGG-decoding tRNA drastically increases error at the AGG codon. The Fluc activity relative to wild type is shown for the negative control (UUU, Phe) and the two Arg codons, AGA and AGG. The black columns represent the activity of the three mutants in the wild-type background and the white columns represent the activity in the background deleted for the tRNA gene. The data are represented as in Figure 3.

FIGURE 6.

Near- and noncognate mutations for histidine-245 (H245). The wild-type, near-cognate, and synonymous noncognate mutant codons for H245 are represented as in Figure 2.

FIGURE 7.

Variation in Fluc activity of H245 mutants. The graph shows the variation in Fluc activity of the near-cognate H245 mutants using the format of Figure 3 except that the y-axis is logarithmic to encompass data over three orders of magnitude. The black columns represent activities that are not significantly different among synonymous codons. The single white column indicates that the activity of the CGC mutant is significantly higher than the synonymous CGU mutant.

FIGURE 8.

Paromomycin increases inaccuracy of only three K529 mutations in S. cerevisiae. The activity of the near-cognate mutants of K529 is shown in the absence (black) or presence (white) of 200 μg/mL paromomycin. Significant increases in activity (ANOVA, P <0.05) were seen for the two nonsense mutants, UAA and UAG, and the Asn mutant, AAU. The data are represented as in Figure 3.

FIGURE 9.

Paromomycin increases inaccuracy of only three H245 codons in S. cerevisiae. The activity of the near-cognate mutants of H245 is shown in the absence (black) or presence (white) of 200 μg/mL paromomycin. Significant increases in activity (ANOVA, P <0.05) were seen for the two Asp mutants, GAU and GAC, and the Arg mutant, CGC. The data are represented as in Figure 7.

FIGURE 10.

Paromomycin increases inaccuracy of five H245 codons in E. coli. The activity of the near-cognate mutants of H245 is shown in the absence (black) or presence (white) of 200 μg/mL paromomycin. Significant increases in activity (ANOVA, P <0.05) were seen for the two Asp mutants, GAU and GAC, two Arg mutants, CGU and CGC, and the Gln mutant, CAG. The data are represented as in Figure 7.