Evidence that the supE44 mutation of Escherichia coli is an amber suppressor allele of glnX and that it also suppresses ochre and opal nonsense mutations

Abstract

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.

FIG. 1.

Photographs showing increased suppression of the ochre mutation in the lacZ gene of the lacZU118 episome in strain MG1655 harboring supE44 compared to the wild type (WT) after 7 days of incubation.

FIG. 2.

Efficiency of suppression of amber, ochre, and opal luxB mutations in the supE44 strain as well as in the isogenic wild-type strain during the stationary growth phase (A) and the logarithmic growth phase (B) and in the supE44 strain with and without ΔrpoS746::kan in the stationary growth phase (C). Suppression efficiencies were calculated as described in Materials and Methods. The values are averages from three independent experiments. The error bars show the standard errors of the means (SEM).

FIG. 3.

Codon-anticodon pairing in suppressor (supE44⁻) and wild-type (supE44⁺) tRNAs, according to the rules of misreading proposed by Lim and Curran (20). Non-Watson-Crick base pairing is indicated by colors.