Quaternary Structure and Deoxyribonucleic Acid-Binding Properties of the Heme-Dependent, CO-Sensing Transcriptional Regulator PxRcoM

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P](1/2) of 250 +/- 10 nM and an average Hill coefficient, n, of 1.7 +/- 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.

Automated summary

RcoM is a heme-containing transcription factor that can sense CO and is one of the regulators of CO metabolism in bacteria. The structure and DNA-binding properties of RcoM are not well characterized. In this study, it was demonstrated that RcoM-1 from Paraburkholderia xenovorans is a dimer and the heme-binding domain mediates dimerization. Through bioinformatics analysis, it was found that RcoM exists in three different genomic contexts, and a refined consensus DNA-binding sequence for RcoM was proposed. It was also discovered that PxRcoM-1 requires all three repeat sites to cooperatively bind DNA. These findings suggest that RcoM interacts with DNA in a unique manner.