In vivo and in vitro activities of the Escherichia coli sigma54 transcription activator, PspF, and its DNA-binding mutant, PspFDeltaHTH.
Transcription of the phage-shock protein (psp) operon in Escherichia coli is driven by a sigma54 promoter, stimulated by integration host factor and dependent on an upstream, cis-acting sequence and an activator protein, PspF. PspF belongs to the enhancer binding protein family but lacks an N-terminal regulatory domain. Purified PspF is not modified and has an ATPase activity that is increased twofold in the presence of DNA carrying the psp cis-acting sequence. Purified mutant His-tagged PspF that lacks the C-terminal DNA-binding motif has a DNA-independent ATPase activity when present at 30-fold the concentration of the wild-type protein. Both proteins oligomerize in solution in an ATP and DNA-independent manner. The wild-type activator protein, but not the DNA-binding mutant, binds specifically to the cis-acting sequence. Analysis of the sequence protected by PspF demonstrates the presence of two upstream binding sites within the sequence, UAS I and UAS II, which together constitute the psp enhancer. Protection at low protein concentrations is more pronounced and more extensive on a supercoiled DNA than on a linear template. Full expression of the psp operon upon hyperosmotic shock depends on wild-type PspF, but only partially requires the presence of the psp enhancer.
Jovanovic
G
G
Laboratory of Genetics, The Rockefeller University, 1230 York Avenue, New York, NY, 10021, USA.
Rakonjac
J
J
Model
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000235:genetics; D006639:Histidine; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D009997:Osmotic Pressure; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C098954:PspF protein, E coli; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C069816:phage shock protein, Bacteria; C497751:rpoN protein, E coli; D006639:Histidine; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases