Recognition of target DNA and transcription activation by the CO-sensing transcriptional activator CooA.
CooA from Rhodospirillum rubrum is a heme-based CO-sensing transcriptional activator, in which CO acts as a physiological effector. In this study, we examined the mechanism of site-specific recognition and transcriptional activation by CooA by elucidating the transcriptional activator activity of the mutant CooA proteins and the chimeric proteins derived from CRP and CooA and the promoter activity of the mutant promoters. Site-directed mutagenesis has revealed that Arg(177), Gln(178), and Ser(181) on the recognition helix of the helix-turn-helix motif in CooA are responsible for the site-specific recognition. The side chains of these amino acid residues at positions 177, 178, and 181 are believed to be hydrogen bonding to the G:A, T:A, and C:G pairs at positions 2/15, 3/14, and 4/13 in the CooA-dependent promoters to recognize the DNA site for CooA. The properties of the CRP/CooA chimeric proteins constructed in this work suggest that CooA activates transcription by a similar mechanism to that of CRP at Class II CRP-dependent promoters.
Aono
S
S
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa, Nomi-gun, 923-1292, Japan. aono@jaist.ac.jp
Takasaki
H
H
Unno
H
H
Kamiya
T
T
Nakajima
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002248:Carbon Monoxide / Q000378:metabolism*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D017931:DNA Primers; D006420:Hemeproteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D002248:Carbon Monoxide; D004247:DNA