Elucidation of the lesions present in the transcription defectivefitA76 mutant ofEscherichia coli: Implication of phenylalanyl tRNA synthetase subunits as transcription factors

Abstract

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 andfitB). We report here the cloning and molecular characterization of a 2-1 kb DNA fragment which complemented the Ts phenotype of thefitA76 andfitA24 mutants but not that due to thefitB 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 includepheS,pheT andrplT genes. Nucleotide sequencing showed that the fragment contains the entirepheS gene and the N-terminal portion ofpheT. Although these results implied that thefitA andpheS genes could be one and the same, earlier data had ruled out such a possibility. In order to know whether thefitA76 mutation defines a novel allele ofpheS, thepheS region of thefitA76 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 thepheS5 mutant. However, it is shown that thefitA76 mutant is primarily transcription-defective while thepheS5 mutant is primarily translation-defective. These results suggested that thefitA76 mutant might harbour another mutation, in addition topheS5. In this report, we present genetic evidence for a second mutation (namedfit95) in thefitA76 mutant. Thefit95 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.