Transcription Activation by CooA, the CO-sensing Factor fromRhodospirillum rubrum

Abstract

CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator from Rhodospirillum rubrum that binds specific DNA se-quences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are anal-ogous to class II CRP-dependent promoters. In this study, we developed an in vivo CooA reporter system in Escherichia coli and an in vitro transcription assay us-ing RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivo in heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with ␣ subunits lacking their C-terminal domain (␣-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter P cooF in vitro, which indicates that ␣-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not ␣-CTD-truncated RNAP, to P cooF . This facilitated binding provides evi-dence for a direct contact between CooA and ␣-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in ␣-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the ␣-CTD at class II promoters. CooA is a CO-sensing transcription activator from Rhodospi-rillum rubrum, and it activates the expression of the cooFSCTJ and cooMKLXUH operons in response to CO (1–3). These two adjacent operons encode proteins that oxidize CO to CO 2 with concomitant reduction of H ϩ to H 2 and allow growth of this organism on CO as a sole energy source (4 – 6). cooA lies imme-diately 3Ј of the cooFSCTJ operon, and its expression does not depend on CO.