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The methylated form of the Ada protein (me Ada) activates transcription from theEscherichia coli ada ,aidB , andalkA promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit ς70 that affect Ada-activated transcription atalkA . Substitution to alanine of residues K593, K597, and R603 in ς70 region 4 results in decreased Ada-dependent binding of RNA polymerase to thealkA promoter in vitro and impairsalkA transcription both in vivo and in vitro, suggesting that these residues define a determinant forme Ada-ς70 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 ς70 region 4 is involved inme Ada-ς70 interaction at theada andaidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affectme Ada-dependent transcription atada andaidB . Unlike the ς70 amino acids involved in the interaction withme Ada at theada andaidB promoters, K593, K597, and R603 are not conserved in ςS , an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. Whileme Ada is able to promote transcription by the ςS form of RNA polymerase (EςS ) atada andaidB , it fails to do so atalkA . We propose thatme Ada can activate transcription at different promoters by contacting distinct determinants in ς70 region 4 in a manner dependent on the location of the Ada binding site.

journal of bacteriology

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