DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus.
Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatus coded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the -10 and -35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the -10 region was calculated to be 4.8 x 10(-9) M, whereas affinity for the palindrome that spans the -35 region was found to be 2.9 x 10(-9) M. Binding of CrtJ to the bchC promoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizing versus reducing conditions.
Ponnampalam
S N
SN
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Bauer
C E
CE
D001426:Bacterial Proteins; D001429:Bacteriochlorophylls / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D002338:Carotenoids / Q000096:biosynthesis*; D017124:Conserved Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003850:Deoxyribonuclease I; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D005810:Multigene Family; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*
D001426:Bacterial Proteins; D001429:Bacteriochlorophylls; C107183:CrtJ protein, Rhodobacter; D004268:DNA-Binding Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D002338:Carotenoids; D010088:Oxidoreductases; C061969:2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase; D003850:Deoxyribonuclease I