Mechanisms of transcriptional regulation at thepspA promoter of Escherichia coli

This thesis analyzes transcriptional regulation of the E. coli phage shock protein (psp) operon. The operon consists of five genes (pspABCDE) that are under control of RNA polymerase containing the alternative {dollar}sigmasp{lcub}54{rcub}{dollar} factor. In addition, transcription of the operon requires activation by the PspF protein belonging to the family of {dollar}sigmasp{lcub}54{rcub}{dollar}-dependent enhancer-binding proteins (EBP) and is positively regulated by the DNA bending protein, Integration host factor (IHF). Studies presented here show that, unusually for an EBP, PspF protein is constitutively active, that DNA binding increases the activity of PspF {dollar}{lcub}sim{rcub}300{dollar}-fold, and that supercoiling can largely substitute for the requirement for IHF on linear templates. In addition, the upstream activation sequences (UAS) in the pspA promoter region that PspF binds are important for pspA transcription in vitro and in vivo only in the presence of IHF. IHF acts to inhibit PspF-dependent transcription on pspA templates either lacking the UAS sites or where the UAS sites have been moved far upstream. Thus, IHF facilitation of PspF activation requires a particular spacing of the UAS relative to the IHF generated bend and the pspA promoter. Activation by the heterologous EBP {dollar}rm NRsb{lcub}I{rcub}{dollar} at the pspA promoter is inhibited by IHF. Likewise, activation by PspF at the {dollar}rm NRsb{lcub}I{rcub}{dollar}-regulated glnH promoter is also inhibited by IHF, whereas activation by {dollar}rm NRsb{lcub}I{rcub}{dollar} is facilitated. Therefore, I argue that it is the combination of the UAS sites and the specific geometry of the promoter resulting from the sharp IHF generated bend that determines the specificity of activation at the pspA promoter.; PspA, a coiled-coiled protein that does not bind DNA and is encoded by the first gene of the psp operon, inhibits pspA transcription in vivo. Purified PspA is active in inhibiting PspF-dependent pspA transcription in vitro as well. This inhibitory activity is specific to {dollar}sigmasp{lcub}54{rcub}{dollar}-dependent transcription, and appears to target PspF.