In vivo and in vitro activities of the Escherichia coli sigma(54) transcription activator, PspF, and its DNA-binding mutant, PspF Delta HTH

Transcription of the phage-shock protein (psp) operon in Escherichia coli i s driven by a sigma(54) promoter, stimulated by integration host factor and dependent on an upstream, cis-acting sequence and an activator protein, Ps pF. PspF belongs to the enhancer binding protein family but lacks an N-term inal regulatory domain. Purified PspF is not modified and has an ATPase act ivity that is increased twofold in the presence of DNA carrying the psp cis -acting sequence. Purified mutant I-Iis-tagged PspF that lacks the C-termin al DNA-binding motif has a DNA-independent ATPase activity when present at 30-fold the concentration of the wild-type protein. Both proteins oligomeri ze in solution in an ATP and DNA-independent manner. The wild-type activato r protein, but not the DNA-binding mutant, binds specifically to the cis-ac ting sequence. Analysis of the sequence protected by PspF demonstrates the presence of two upstream binding sites within the sequence, UAS I and UAS I I, which together constitute the psp enhancer. Protection at low protein co ncentrations is more pronounced and more extensive on a supercoiled DNA tha n 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. (C) 1999 Academic Press.