MAPPING THE CAMP RECEPTOR PROTEIN CONTACT SITE ON THE ALPHA-SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE

The C-terminal region (amino acid residues 236-329) of the Escherichia coli RNA polymerase alpha-subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C-terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant alpha-subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP-CRP. DNA sequence determination of several 'Lac-' mutant rpoA genes revealed that all had mutations clustered within a short segment near the C-terminus of alpha, between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).