The C-terminal domains of the RNA polymerase α subunits: contact site with fis and localization during co-activation with CRP at the Escherichia coli proP P2 promoter

Fis is a versatile transactivator that functions at many different promoters. Fis activates transcription at the RpoS-dependent proP P2 promoter when bound to a site that overlaps the −35 hexamer by a mechanism that requires the C-terminal domain of the α subunit of RNA polymerase (αCTD). The region on Fis responsible for activating transcription through the αCTD has been localized to a short β-turn near the DNA-binding determinant on one subunit of the Fis homodimer. We report here that Fis-dependent activation of proP P2 transcription requires two discrete regions on the αCTD. One region, consisting of residues 264–265 and 296–297, mediates DNA binding. A second patch, comprising amino acid residues 271–273, forms a ridge on the surface of the αCTD that we propose interacts with Fis. The accompanying paper shows that these same regions on αCTD are utilized for transcriptional activation at the rrnB and rrnE P1 promoters by Fis bound to a site upstream of the core promoter (centered at −71/−72). In addition to stimulation of proP P2 transcription by Fis, CRP co-activates this promoter when bound to a remote site upstream from the promoter (centered at −121.5). RNA polymerase preparations lacking one αCTD of the α dimer were employed to demonstrate that the β′-associated αIICTD was utilized preferentially by Fis at proP P2 in the presence and absence of CRP. These experiments define the overall architecture of the proP P2 initiation complex where Fis and CRP each function through a different αCTD.