Journal of Bacteriology, March 1999, p. 1524-1529, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
70 Subunit of RNA
Polymerase According to Promoter Architecture: Identification of
the Target of Ada Activation at the alkA
Promoter
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Received 28 September 1998/Accepted 18 December 1998
The methylated form of the Ada protein (meAda)
activates transcription from the Escherichia coli ada,
aidB, and alkA promoters with different
mechanisms. In this study we identify amino acid substitutions in
region 4 of the RNA polymerase subunit
70 that affect
Ada-activated transcription at alkA. Substitution to
alanine of residues K593, K597, and R603 in
70 region 4 results in decreased Ada-dependent binding of RNA polymerase to the
alkA promoter in vitro and impairs alkA
transcription both in vivo and in vitro, suggesting that these residues
define a determinant for meAda-
70
interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem.
273:13307-13312, 1998), we showed that a set of negatively charged
amino acids in
70 region 4 is involved in
meAda-
70 interaction at the ada
and aidB promoters. However, the alanine substitutions of
positively charged residues K593, K597, and R603 do not affect
meAda-dependent transcription at ada and
aidB. Unlike the
70 amino acids involved in
the interaction with meAda at the ada and
aidB promoters, K593, K597, and R603 are not conserved in
S, an alternative
subunit of RNA polymerase mainly
expressed during the stationary phase of growth. While
meAda is able to promote transcription by the
S form of RNA polymerase (E
S) at
ada and aidB, it fails to do so at
alkA. We propose that meAda can activate
transcription at different promoters by contacting distinct
determinants in
70 region 4 in a manner dependent on the
location of the Ada binding site.
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