MAPPING OF THE OXYR PROTEIN CONTACT SITE IN THE C-TERMINAL REGION OF RNA-POLYMERASE ALPHA-SUBUNIT

The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase alpha subunit for cooperative interact ion,vith and transcription activation at OxyR-dependent promoters, sug gesting direct protein-protein contact between OxyR and the C-terminal region of the alpha subunit, To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutationa l analysis of the 3' half of E. coli rpoA, the gene encoding the alpha subunit of RNA polymerase. We isolated a number of rpoA mutants defec tive in oxyR-dependent transcription activation at the E. coli katG pr omoter, Nucleotide sequence analysis of the rpoA gene from these mutan ts revealed that the mutations showing clear phenotypes are all cluste red at two narrow regions (amino acid residues 265 to 269 aad 293 to 3 00) within the C terminus of the alpha subunit. Reconstituted RNA poly merases containing the mutant alpha subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promote rs by OxyR, These results suggest that these two regions comprise the contact surfaces on the alpha subunit for OxyR.