Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli.

Abstract

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major or-factor r 7~ To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes. We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties. The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of ~r 7~ Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most. The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters. Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes. These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB.

Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent

“…Thus, it is possible that the role of PhoP phosphorylation is to favour protein-protein interactions with the transcriptional machinery. This possibility is supported by previous studies showing that PhoB interacts with s 70 (Kumar et al, 1994;Makino et al, 1993), and that OmpR interacts with the C-terminal domain of the RNA polymerase a subunit (Slauch et al, 1991).…”

Regulation and Properties of PstSCAB, a High-Affinity, High-Velocity Phosphate Transport System ofSinorhizobium meliloti

“…PstC and PstA are annotated as phosphate transport permeases, and PstB is the transport ATP binding protein. PhoU is predicted to be involved in regulation, and PhoB is the response regulator believed to be activated by phosphorylation by its cognate sensor protein PhoR (40,41,42,65).…”

σ70 and PhoB activator

“…Existing biochemical data suggest that the recruitment of the RNAPH does not take place unless PhoB is bound to the promoter first. 20 The DNA binding sequence in the pho box differs from the consensus sequence in the -35 element and thus the σ 4 domain of σ 70 requires a previous PhoB binding to stabilize its own binding to the DNA.…”