Molecular determinants for PspA-mediated repression of the AAA transcriptional activator PspF.
The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the sigma(54) subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of sigma(54) AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal alpha-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.
Elderkin
Sarah
S
Imperial College London, Department of Biological Sciences, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, UK.
Bordes
Patricia
P
Jones
Susan
S
Rappas
Mathieu
M
Buck
Martin
M
D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D007763:Lac Operon; D008958:Models, Molecular; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C098954:PspF protein, E coli; D015534:Trans-Activators; C069816:phage shock protein, Bacteria; D001616:beta-Galactosidase