The frequency of translational misreading errors in E. coli is largely determined by tRNA competition - PubMed (original) (raw)

The frequency of translational misreading errors in E. coli is largely determined by tRNA competition

Emily B Kramer et al. RNA. 2007 Jan.

Abstract

Estimates of missense error rates (misreading) during protein synthesis vary from 10(-3) to 10(-4) per codon. The experiments reporting these rates have measured several distinct errors using several methods and reporter systems. Variation in reported rates may reflect real differences in rates among the errors tested or in sensitivity of the reporter systems. To develop a more accurate understanding of the range of error rates, we developed a system to quantify the frequency of every possible misreading error at a defined codon in Escherichia coli. This system uses an essential lysine in the active site of firefly luciferase. Mutations in Lys529 result in up to a 1600-fold reduction in activity, but the phenotype varies with amino acid. We hypothesized that residual activity of some of the mutant genes might result from misreading of the mutant codons by tRNA(Lys) (UUUU), the cognate tRNA for the lysine codons, AAA and AAG. Our data validate this hypothesis and reveal details about relative missense error rates of near-cognate codons. The error rates in E. coli do, in fact, vary widely. One source of variation is the effect of competition by cognate tRNAs for the mutant codons; higher error frequencies result from lower competition from low-abundance tRNAs. We also used the system to study the effect of ribosomal protein mutations known to affect error rates and the effect of error-inducing antibiotics, finding that they affect misreading on only a subset of near-cognate codons and that their effect may be less general than previously thought.

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Figures

FIGURE 1.

FIGURE 1.

Near- and noncognate mutations for Lysine-529. 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 synonymous codons, differing in more than one codon position, are shown in black on gray. The control UUU codon is shown in boldface.

FIGURE 2.

FIGURE 2.

Variation in F-luc activity of K5290 mutants. The graph shows the F-luc expression of the indicated constructs expressed as a fraction of the expression of wild-type F-luc. The mutants include a deletion of the entire firefly luc gene (Fluc) and those replacing the K529 codon (AAA) with the indicated codon. Indicated below each codon is the amino acid it encodes. Error bars are standard errors of the mean.

FIGURE 3.

FIGURE 3.

High residual F-luc activity of some K529 codons results from near-cognate decoding. A comparison of the F-luc activity relative to wild type of synonymous near- and noncognate codon mutants. Codon identity is shown as in Figure 2.

FIGURE 4.

FIGURE 4.

Overexpression of tRNAArg UCU reduces misreading errors at Arg codons. The F-luc activity relative to wild type is shown for mutants replacing the K529 codon with the Arg codons AGA or AGG. Black bars indicate expression in a wild-type genetic background (WT) and gray bars indicate expression in the presence of overexpressed tRNAArg UCU.

FIGURE 5.

FIGURE 5.

Aminoglycoside antibiotics increase misreading on a subset of near-cognate codons. The F-luc activities relative to wild type are shown for mutants carrying the indicated K529 codon replacements in the presence of no antibiotic (black bars), 5 μg/mL paromomycin (gray bars), or 2 μg/mL streptomycin (white bars).

FIGURE 6.

FIGURE 6.

A mutation in rpsD increases misreading only of error-prone codons. The F-luc activities relative to wild type of the indicated K529 codon replacements are shown from a wild-type strain (black bars) and a strain carrying the rpsD12 mutation (gray bars).

FIGURE 7.

FIGURE 7.

A mutation in rpsL reduces misreading only of error-prone codons. The F-luc activities relative to wild type of the indicated K529 codon replacements are shown from a wild-type strain (black bars) and a strain carrying the rpsL141 mutation (gray bars).

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