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.

Fig. 1

Amino acid sequence comparison of the C-terminal domains of PrrA homologues and E. coli Fis. Amino acids are aligned according to the structure of PrrA-CTD (t indicates the two turns, one before helix 1 and one between helices 1 and 2; Laguri et al., 2003). Amino acids shaded are shared among PrrA homologues (Elsen et al., 2004). Rs, R. sphaeroides 2.4.1 (GenBank accession no. AAA86723); Rc, Rhodobacter capsulatus B10 (P42508); Rd, Roseobacter denitrificans OCh114 (AB010723); Bj, Bradyrhizobium japonicum 110spc4 (CAA06859); Pa, Pseudomonas aeruginosa PA01 (AAT50683); Ec, E. coli MG1655 Fis (P0A6R3).

Fig. 2

In vitro transcription assays using the PrrA-dependent cycA P2 promoter. The cycA P2 promoter fragment extends from −73 to +22 relative to the transcription start site (Karls et al., 1999). The RNA1 transcript (~108 nt) was produced by the PrrA-independent oriV promoter. The concentration of acetyl phosphate-treated wild-type (WT) or mutant PrrA protein is indicated above the panel. Numbers below each lane report the relative amount of cycA P2 transcript (~176 nt) produced after correction for background and normalization to the amount of RNA1 transcript. nd, None detected.

Fig. 3

In vitro transcription assays using the PrrA-dependent puc promoter. The puc promoter fragment extends from −629 to +33 relative to the transcription initiation site (Lee & Kaplan, 1992). The RNA1 transcript (~108 nt) was produced by the PrrA-independent oriV promoter. The concentration of acetyl phosphate-treated wild-type (WT) or mutant PrrA protein is indicated above the panel. Numbers below each lane report the relative amount of puc transcript (~187 nt) produced after correction for background and normalization to the amount of RNA1 transcript.

Fig. 4

DNase I protection of the PrrA-dependent cycA P2 promoter by wild-type or mutant PrrA proteins. Indicated concentrations of acetyl phosphate-treated wild-type or mutant PrrA proteins (0, 0·5, 1, 2 or 4 µM) were incubated with end-labelled cycA P2 promoter DNA and treated with DNase I. Hatched bars represent the region of cycA P2 DNA protected by wild-type or mutant PrrA proteins.