Elucidation of the lesions present in the transcription defective fitA76 mutant of Escherichia coli: Implication of phenylalanyl tRNA synthetase subunits as transcription factors

Earlier reports from our laboratory dealt with the identification, mapping and characterization of a temperature sensitive mutant (fitA76) with a primary transcription defect at 42° C and two of its suppressors (fitA24 and fitB). We report here the cloning and molecular characterization of a 2·1 kb DNA fragment which complemented the Ts phenotype of the fitA76 and fitA24 mutants but not that due to the fitB mutant. Cloning of this fragment in the T7 expression vector pT7·5 revealed the synthesis of a 33 kDa protein. The fragment hybridized with the Kohara phages 322 and 323 whose overlapping regions include pheS, pheT and rplT genes. Nucleotide sequencing showed that the fragment contains the entire pheS gene and the N-terminal portion of pheT. Although these results implied that the fitA and pheS genes could be one and the same, earlier data had ruled out such a possibility. In order to know whether the fitA76 mutation defines a novel allele of pheS, the pheS region of the fitA76 mutant was also sequenced, revealing a G ® A nucleotide transition at position 293 of the coding region. This lesion is the same as that reported for the pheS5 mutant. However, it is shown that the fitA76 mutant is primarily transcription-defective while the pheS5 mutant is primarily translation-defective. These results suggested that the fitA76 mutant might harbour another mutation, in addition to pheS5. In this report, we present genetic evidence for a second mutation (named fit95) in the fitA76 mutant. The fit95 by itself confers a Ts phenotype on rich media devoid of sodium chloride. It is proposed that the subunits of phenylalanyl tRNA synthetase could act as transcription factors (Fit) also.