Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA

Abstract

The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced <45 % as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.

Postgenomic Adventures with Rhodobacter sphaeroides

“…The fold of this binding domain is a Fis-type fold. In addition, in order to bind DNA in a specific manner, the protein must be phosphorylated (52,58), which results in its dimerization (57,58) and subsequent occupation of the two DNA half-sites.…”

Development of the bacterial photosynthetic apparatus

“…The emerging picture predicts that the PrrBA functions in a homeostatic feedback loop to balance the production of reducing power from light or other resources (when O 2 is limiting) with induction of synthesis pathways that recycle excess reductant. The transcription factor in this pathway (PrrA/RegA) binds numerous promoters and activates transcription in vitro [4], its C-terminus contains a DNA binding motif from the Fis family of transcription factors [31,32], and target sites for this protein are predicted to exist upstream of numerous other, uncharacterized genes [7,8•]. Like many response regulators, PrrA activity increases upon phosphorylation [33], presumably because this stimulates PrrA dimerization [34], which is needed to activate transcription [35].…”

Hierarchical Binding of the TodT Response Regulator to Its Multiple Recognition Sites at the tod Pathway Operon Promoter

“…7,8 Among the processes regulated by TCS in bacteria are host invasion, drug resistance, Mg 2+ homeostasis, motility, phosphate uptake, carbon utilization (cellobiose, phenol, etc. ), osmoregulation, and nitrogen fixation.…”