Binding site determinants for the LysR-type transcriptional regulator PcaQ in the legume endosymbiont Sinorhizobium meliloti.
LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the beta-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions -78 to -45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many alpha-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.
Center for Environmental Genomics, Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Allyson M
AM
MacLean
Michelle I
MI
Anstey
Turlough M
TM
Finan
D016960:Agrobacterium tumefaciens / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016172:DNA Fingerprinting; D024202:Electrophoretic Mobility Shift Assay; D007887:Fabaceae / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D042163:Rhizobium etli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators