Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6/(Leu)

Abstract

Escherichia coli has only a single copy of a gene for tRNA6/(Leu) (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4/(Leu), which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl- O2-methyluridine) as its anticodon, tRNA6/(Leu) is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6/(Leu) with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature- sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6/(Leu). These tRNA6/(Leu)-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4/(Leu) restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4/(Leu) poorly recognizes the UUG codon at 42°C and that the wild-type tRNA6/(Leu) is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6/(Leu) remains to be investigated.