Single transduction in the transcriptional activator CooA containing a heme-based CO sensor: isolation of a dominant positive mutant which is active as the transcriptional activator even in the absence of CO.
We constructed an in vivo reporter system to measure the activity of CooA as the transcriptional activator and showed that the recombinant CooA was active as the transcriptional activator in the presence of CO even in E. coli cells. A dominant positive mutant of CooA, in which Met131 was replaced by Leu, was isolated by a random mutagenesis with this reporter system. The electronic absorption spectra of M131L mutant were identical to those of wild type CooA in oxidized (Fe3+), reduced (Fe2+), and CO-bound (CO-Fe2+) state, indicating that the coordination structure and environment of the heme were not changed by this mutation. Methionine at position 131 was the carboxyl-terminal end of the heme-binding domain of CooA, which would be adjacent to the hinge region connecting the heme-binding domain and the DNA-binding domain.
Aono
S
S
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan. aono@jaist.ac.jp
Matsuo
T
T
Shimono
T
T
Ohkubo
K
K
Takasaki
H
H
Nakajima
H
H
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013053:Spectrophotometry; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D006418:Heme; D002248:Carbon Monoxide; D001616:beta-Galactosidase