NOVEL TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA-COLI THAT ARE UNABLE TO GROW IN THE ABSENCE OF WILD-TYPE TRNA(6)(LEU)

Escherichia coli has only a single copy of a gene for tRNA(6)(Leu) (Y, Komine et al,, J, Mel. Biol, 212:579-598, 1990). The anticodon of thi s tRNA is CAA (the wobble position C is modified to O-2-methylcytidine ), and it recognizes the codon UUG, Since UUG is also recognized by tR NA(4)(Leu), which has UAA (the wobble position U is modified to 5-carb oxymethylaminomethyl-O-2-methyluridine) as its anticodon, tRNA(6)(Leu) is not essential for protein synthesis. The BT63 strain has a mutatio n in the anticodon of tRNA(6)(Leu) with a change from CAA to CUA, whic h results in the amber suppressor activity of this strain (supP, Su(+) 6), We isolated 18 temperature-sensitive (ts) mutants of the BT63 stra in whose temperature sensitivity was complemented by introduction of t he wild-type gene for tRNA(6)(Leu). These tRNA(6)(Leu)-requiring mutan ts were classified into two groups. The 10 group I mutants had a mutat ion in the miaA gene, whose product is involved in a modification of t RNAs that stabilizes codon-anticodon interactions. Overexpression of t he gene for tRNA(4)(Leu) restored the growth of group I mutants at 42 degrees C, Replacement of the CUG codon with UUG reduced the efficienc y of translation in group I mutants. These results suggest that unmodi fied tRNA(4)(Leu) poorly recognizes the UUG codon at 42 degrees C and that the wild-type tRNA(6)(Leu) is required for translation in order t o maintain cell viability. The mutations in the six group II mutants w ere complemented by introduction of the gidA gene, which may be involv ed in cell division. The reduced efficiency of translation caused by r eplacement of the CUG codon with UUG was also observed in group II mut ants. The mechanism of requirement for tRNA(6)(Leu) remains to be inve stigated.