ROLE OF UPSTREAM ACTIVATION SEQUENCES AND INTEGRATION HOST FACTOR IN TRANSCRIPTIONAL ACTIVATION BY THE CONSTITUTIVELY ACTIVE PROKARYOTIC ENHANCER-BINDING PROTEIN PSPF

PspF, the transcriptional activator of the pspA operon of Escherichia coli, which belongs to the enhancer binding protein (EBP) family of si gma(54) activator proteins, is constitutively active in an in vitro tr anscription assay. PspF protein, together with RNA polymerase holoenzy me containing sigma(54), is required for in vitro transcription from t he pspA promoter. EBP proteins are typically subject to regulation eit her by post-translational modification or interaction of a specific li gand with an N-terminal regulatory domain. However, unlike other membe rs of the EBP family, PspF lacks this domain. pspA is positively regul ated by IHF in vitro, and this regulation is dependent on the topology of the DNA; a linear template is much more dependent on IHF than a su percoiled template. EBP binding to upstream activating sequences (UAS) in their target promoters is mediated by the C-terminal domain which contains a helix-turn-helix DNA-binding motif. A mutant PspF protein l acking the C-terminal DNA-binding domain is active in vitro, although at much higher concentrations than the wild-type protein. In vitro tra nscription from pspA templates missing one or both of the UAS sites is reduced relative to wild-type templates, but is still appreciable; ho wever, IHF acts as a negative regulator of pspA transcription on these mutant templates. Thus, PspF bound to non-specific sequences upstream of the pspA promoter can activate pspA transcription, but this activa tion is inhibited by IHF. These data, taken together, support the mode l that a precise promoter geometry is necessary for IHF to positively regulate transcription and that IHF may act to prevent activation from inappropriately spaced upstream sites. (C) 1997 Academic Press Limite d.