Mapping of the OxyR Protein Contact Site in the C-Terminal Region of RNA Polymerase (cid:97) Subunit

The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase (cid:97) subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the (cid:97) subunit. To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3 (cid:42) half of E. coli rpoA , the gene encoding the (cid:97) subunit of RNA polymerase. We isolated a number of rpoA mutants defective in oxyR -dependent transcription activation at the E. coli katG promoter. Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the (cid:97) subunit. Reconstituted RNA polymerases containing the mutant (cid:97) subunits were unable to respond to transcription activation in vitro at the katG , ahpC , and oxyX promoters by OxyR. These results suggest that these two regions comprise the contact surfaces on the (cid:97) subunit for OxyR. Several lines of evidence indicate that transcription activa- tion by DNA-binding transcription factors involves a direct protein-protein contact between those factors and RNA poly- merase (1, 13). With RNA polymerases containing mutant (cid:97) subunits with C-terminal truncation, Effect of mutations on UP protein(s). In our systematic mapping of the contact site I region on the (cid:97) subunit, we isolated in this study a set of rpoA mutants defective in OxyR-dependent transcription by random mu- tagenesis of the C-terminal proximal half of the rpoA gene followed by in vivo screening for decreased expression of lacZ under the control of the OxyR-regulated katG promoter. The mutations showing the clear mutant phenotype are clustered at two regions, around amino acid residues 268 to 269 (and 265) and 298 to 299. Mutations with less profound phenotypes were also obtained in the vicinity of the downstream contact site, including residues 293, 294, 300, and 307. The results of in vitro transcription experiments with the reconstituted RNA polymerases containing these mutant (cid:97) subunits are generally in good agreement with the in vivo data. Thus, we concluded that the contact site for OxyR is composed of the two short segments, one residues 265 and 269 and the other be- tween residues 293 and 300. These two segments forming the OxyR contact surface are both located on the hydrophilic side of helix-1 and helix-4, respectively, as analyzed by multidimen-sional heteronuclear magnetic resonance