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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.

The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the sigma 70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkA Promoter

Paolo Landini* and Stephen J. W. Busby

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 sigma 70 that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in sigma 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-sigma 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 sigma 70 region 4 is involved in meAda-sigma 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 sigma 70 amino acids involved in the interaction with meAda at the ada and aidB promoters, K593, K597, and R603 are not conserved in sigma S, an alternative sigma  subunit of RNA polymerase mainly expressed during the stationary phase of growth. While meAda is able to promote transcription by the sigma S form of RNA polymerase (Esigma 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 sigma 70 region 4 in a manner dependent on the location of the Ada binding site.

* Corresponding author. Mailing address: School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44-121-414-5434. Fax: 44-121-414-7366. E-mail: landini{at}eawag.ch.

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.


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