The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter
ABSTRACT 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 ς70 that affect Ada-activated transcription at alkA. 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 impairs alkAtranscription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-ς70interaction. 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 inmeAda-ς70 interaction at the adaand aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affectmeAda-dependent transcription at ada andaidB. Unlike the ς70 amino acids involved in the interaction with meAda at the ada 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. WhilemeAda is able to promote transcription by the ςS form of RNA polymerase (EςS) atada and aidB, it fails to do so atalkA. We propose that meAda 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.