A Negative Element
Located in the Upstream Flanking Region of the Gene Encoding Arginyl-tRNA
Synthetase (argS) from Escherichia
coli
LIU Mo-Fang, XU Min-Gang, XIA Xian, WANG En-Duo*,
WANG Ying-Lai
( State Key Laboratory of Molecular Biology, Institute of Biochemistry and
Cell Biolog, Shanghai Institutes for Biological Sciences, the Chinese Academy
of Sciences, Shanghai 200031, China )
Abstract 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 (argSΔ1) or the region
in front of Shine-Dalgarno Sequence (argSΔ2) or the -10 region of
promoter (argSΔ3), 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 (argSΔ4), the -35 region (argSΔ5), -52 nt
(argSΔ6), -70 nt (argSΔ7) and -122 nt (argSΔ8),
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 argSΔ4,argSΔ5,
argSΔ6, some of ArgRS formed an inclusion body. By determination of
RNA dot hybridization, the amount of mRNA produced in the transcription of argSΔ4,
argSΔ5 and argSΔ6 was about 2–3 times than that of the wild
type argS, argSΔ7 and argSΔ8. 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.
Key words arginyl-tRNA synthetase； negative regulation； Escherichia coli
*Corresponding author： Tel, 86-21-64374430； Fax, 86-21-64338357； e-mail, [email protected]