posted on 2022-04-08, 17:10authored byMatthew
R. Dent, Madeleine G. Roberts, Hannah E. Bowman, Brian R. Weaver, Darrell R. McCaslin, Judith N. Burstyn
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.