The C-terminal domains of the RNA polymerase alpha 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 minus sign35 hexamer by a mechanism that requires the C-terminal domain of the alpha subunit of RNA polymerase (alphaCTD). The region on Fis responsible for activating transcription through the alphaCTD has been localized to a short beta-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 alphaCTD. 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 alphaCTD that we propose interacts with Fis. The accompanying paper shows that these same regions on alphaCTD 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 minus sign71/minus sign72). 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 alphaCTD of the alpha dimer were employed to demonstrate that the beta'-associated alpha(II)CTD 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 alphaCTD.
McLeod
Sarah M
SM
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095-1737, USA.
Aiyar
Sarah E
SE
Gourse
Richard L
RL
Johnson
Reid C
RC
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D035021:Factor For Inversion Stimulation Protein; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D034982:Integration Host Factors; D008958:Models, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D027981:Symporters / Q000235:genetics*; D015533:Transcriptional Activation
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; C079244:ProP protein, E coli; D021122:Protein Subunits; D027981:Symporters; C440502:integration host factor, E coli; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit