Determinants of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit important for transcription at class I cyclic AMP receptor protein-dependent promoters.

Abstract

Alanine scanning of the Escherichia coli RNA polymerase alpha subunit C-terminal domain (alphaCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of alphaCTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for alphaCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in alphaCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.