Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase

Abstract

The genetic code is established in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases. Many aminoacyl-tRNA synthetases require an additional domain for editing, to correct errors made by the catalytic domain. A nonfunctional editing domain results in an ambiguous genetic code, where a single codon is not translated as a specific amino acid but rather as a statistical distribution of amino acids. Here, wide-ranging consequences of genetic code ambiguity in Escherichia coli were investigated with an editing-defective isoleucyl-tRNA synthetase. Ambiguity retarded cell growth at most temperatures in rich and minimal media. These growth rate differences were seen regardless of the carbon source. Inclusion of an amino acid analogue that is misactivated (and not cleared) diminished growth rate by up to 100-fold relative to an isogenic strain with normal editing function. Experiments with target-specific antibiotics for ribosomes, DNA replication, and cell wall biosynthesis, in conjunction with measurements of mutation frequencies, were consistent with global changes in protein function caused by errors of translation and not editing-induced mutational errors. Thus, a single defective editing domain caused translationally generated global effects on protein functions that, in turn, provide powerful selective pressures for maintenance of editing by aminoacyl-tRNA synthetases.

Fig. 1.

Effect of temperature on the growth rate of editing-deficient and wild-type strains. Growth curves of the strains were generated in a microplate reader. Data were fitted to the logistic growth equation (see Materials and Methods) to generate an intrinsic growth rate (a growth rate of 1 h^–1 corresponds to a doubling time of ≈42 min). Strains were tested in rich media (LB) (A) and minimal media (MSglc) (B). Only at the lowest temperatures tested do strains have comparable growth rates. Error bars give the standard deviation of the growth rates of three replicates of each of three clones.

Fig. 2.

Effect of Nrv concentration on growth rate. Growth rates were determined for editing-deficient and wild-type strains over 4 orders of magnitude of Nrv concentration (Left). A 2-order-of-magnitude effect was seen between editing-deficient and wild-type strains. Error bars indicate the standard deviations of the growth rates of three replicates of each of three clones. In addition, halo assays testing for sensitivity to Nrv showed a 2.5-fold difference in clearing zone area (Right). Error bars give the standard deviations of the clearing zones of three clones.

Fig. 3.

Growth halo test of antibiotics. Editing-deficient and wild-type strains were tested for sensitivity to antibiotics by diffusion in halo tests (see Materials and Methods). The diameters of the clearing zones caused by the antibiotics were measured, and the area of the clearing zone was calculated. In all cases, the editing-deficient strain was found to be more sensitive to antibiotics. Error bars give the standard deviations of the clearing zones of three clones.

Fig. 4.

Mutation rate effects on editing-deficient and wild-type strains. (A) Halo tests of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and 2-aminopurine (2AP). MNNG may induce slightly greater clearing zone in the wild-type strain. Error bars give the standard deviations of the clearing zones of three clones. (B) Frequencies of spontaneous mutants resistant to rifampicin (Rif^R) and Nal (Nal^R). Strains tested included the editing-deficient and wild-type strains; ΔdnaQ, mutator versions of these two strains; and two MG1655-derived strains with, separately, ΔdnaQ and ΔmutS (to induce high-mutation frequencies). Mutator strains have greatly enhanced mutation frequencies, whereas editing-deficient and wild-type strains are highly comparable. Error bars give standard deviations of mutation frequencies of three clones.