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 in^meAda-ς⁷⁰ interaction at the adaand aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect^meAda-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 ς^S, an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. While^meAda is able to promote transcription by the ς^S form of RNA polymerase (Eς^S) 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.