A Negative Element Located in the Upstream Flanking Region of the Gene Encoding Arginyl-tRNA Synthetase (argS) from...

The gene, argS, encoding the arginyl-tRNA synthetase (ArgRS) from Escherichia coli ( E.coli ) was overexpressed 1 000 fold in the transformant when E. coli TG1 was transformed with the recombinant plasmid containing argS and pUC18. In order to investigate the regulation of expression of E. coli argS, a series of deletion mutations was constructed. The results of SDS-PAGE showed that deletions of the whole 5' flanking region (argSdelta1) or the region in front of Shine-Dalgarno Sequence (argSdelta2) or the -10 region of promoter (argSdelta3), caused no overexpression of argS. If argS was deleted from 3' end of the flanking region (-189 nt) to the upstream of -10 region of promoter (argSdelta4), the -35 region (argSdelta5), -52 nt (argSdelta6), -70 nt (argSdelta7) and -122 nt (argSdelta8), respectively, the mutant gene was overexpressed to a level similar to that of argS bringing the full length 5' flanking region. However, in the expression of argSdelta4, argSdelta5, argSdelta6, some of ArgRS formed an inclusion body. By determination of RNA dot hybridization, the amount of mRNA produced in the transcription of argSdelta4, argSdelta5 and argSdelta6 was about 2--3 times than that of the wild type argS, argS delta7 and argS delta8. This indicated that the deletion of a 19 nt sequence (AATAGTGAAAACGGCAATA) located between -52 nt and -70 nt of the gene increased the transcription of argS. The 19 nt sequence is a negative region that represses transcription of argS. Deletion of the negative element may result in a faster production of ArgRS and the accumulation of some unfolding protein intermediates aggregating to form the inclusion body. The result by analysis of gel retardation shows that a factor binds to the negative element. Arginine induced specifically the transcription of argS and its effect correlated with the above negative element.