Localization of amino acids required for Fis to function as a class II transcriptional activator at the RpoS-dependent proP P2 promoter.
ProP is an integral membrane transporter of proline, glycine betaine, and several other osmoprotecting compounds. Fis plus RpoS collaborate to promote a burst of proP transcription in late exponential growth phase. This brief period of ProP synthesis enables stationary phase cells to cope with a potential hyperosmotic shock. Fis activates the RpoS (sigma(38))-dependent proP P2 promoter by binding to a site within the promoter region centered at -41 and thus functions as a class II activator. We show here that activation by Fis at this promoter is completely dependent upon the alpha-CTD of RNA polymerase and that the activation domain on Fis is localized to a four amino acid ridge on the surface of Fis adjacent to the helix-turn-helix DNA binding domain in only one subunit of the homodimer. Fis mutants containing amino acid substitutions within this region are defective in cooperative binding interactions with the sigma(38)-form of RNA polymerase. Some of these substitutions also alter interactions with DNA sequences flanking the core binding site, but we show that changes in Fis-mediated curvature do not affect promoter activity. We conclude that the same amino acids are used by Fis to activate transcription from a class I (-71, rrnB P1) and class II (-41, proP P2) location, but this region is distinct from that required to regulate the Hin site-specific DNA inversion reaction.
McLeod
S M
SM
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095-1737, USA.
Xu
J
J
Cramton
S E
SE
Gaal
T
T
Gourse
R L
RL
Johnson
R C
RC
D001120:Arginine; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D008958:Models, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D027981:Symporters; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; C079244:ProP protein, E coli; D012808:Sigma Factor; D027981:Symporters; D015534:Trans-Activators; C069567:sigma factor KatF protein, Bacteria; D001120:Arginine; C106402:RNA polymerase Esigma(38); D012321:DNA-Directed RNA Polymerases