Nitric oxide-responsive interdomain regulation targets the σ54-interaction surface in the enhancer binding protein NorR.
Bacterial enhancer binding proteins (bEBPs) are specialized transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ(54). Transcriptional activation by the NorR bEBP is controlled by a regulatory GAF domain that represses the ATPase activity of the central AAA+ domain in the absence of nitric oxide. Here, we investigate the mechanism of interdomain repression in NorR by characterizing substitutions in the AAA+ domain that bypass repression by the regulatory domain. Most of these substitutions are located in the vicinity of the surface-exposed loops that engage σ(54) during the ATP hydrolysis cycle or in the highly conserved GAFTGA motif that directly contacts σ(54). Biochemical studies suggest that the bypass mutations in the GAFTGA loop do not influence the DNA binding properties of NorR or the assembly of higher order oligomers in the presence of enhancer DNA, and as expected these variants retain the ability to activate open complex formation in vitro. We identify a crucial arginine residue in the GAF domain that is essential for interdomain repression and demonstrate that hydrophobic substitutions at this position suppress the bypass phenotype of the GAFTGA substitutions. These observations suggest a novel mechanism for negative regulation in bEBPs in which the GAF domain targets the σ(54)-interaction surface to prevent access of the AAA+ domain to the sigma factor.
Bush
Matthew
M
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney NR4 7UH, UK.
Ghosh
Tamaswati
T
Tucker
Nicholas
N
Zhang
Xiaodong
X
Dixon
Ray
R
D019943:Amino Acid Substitution; D004269:DNA, Bacterial / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D055503:Protein Multimerization; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C504602:NorR protein, E coli; D015534:Trans-Activators; D009569:Nitric Oxide; D051877:RNA Polymerase Sigma 54