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

The methylated form of the Ada protein ((me)Ada) 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 ς(70) that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in ς(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 (me)Ada-ς(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 ς(70) region 4 is involved in (me)Ada-ς(70) interaction at the ada and aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect (me)Ada-dependent transcription at ada and aidB. Unlike the ς(70) amino acids involved in the interaction with (me)Ada at the ada and aidB promoters, K593, K597, and R603 are not conserved in ς(S), an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. While (me)Ada is able to promote transcription by the ς(S) form of RNA polymerase (Eς(S)) at ada and aidB, it fails to do so at alkA. We propose that (me)Ada can activate transcription at different promoters by contacting distinct determinants in ς(70) region 4 in a manner dependent on the location of the Ada binding site.