Half-site DNA sequence and spacing length contributions to PrrA binding to PrrA site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1

The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.