Mapping CooA center dot RNA polymerase interactions - Identification of activating regions 2 and 3 in CooA, the co-sensing transcriptional activator

CooA is a CO-sensing protein that activates the transcription of genes enco ding the CO-oxidation (coo) regulon, whose polypeptide products are require d for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P-cooF promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumara te and nitrate reductase activator protein)-dependent promoters when expres sed in Escherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion of cooA encoding the effector-bi nding domain. Some of the mutations affect regions of CooA that are homolog ous to the activating regions (AR2 and AR3) previously identified in CRP an d FNR, whereas others affect residues that lie in a region of CooA between AR2 and ARS. These CooA variants are comparable to wild-type (WT) CooA in D NA binding affinity in response to CO but differ in transcription activatio n, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA va riants were obtained in the AR2 and AR3 regions that have minimal transcrip tional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2- and AR3-like surfaces t hat are required for optimal transcription activation.