DNA-BINDING CHARACTERISTICS OF CRTJ - A REDOX-RESPONDING REPRESSOR OFBACTERIOCHLOROPHYLL, CAROTENOID, AND LIGHT HARVESTING-II GENE-EXPRESSION IN RHODOBACTER-CAPSULATUS
Sn. Ponnampalam et Ce. Bauer, DNA-BINDING CHARACTERISTICS OF CRTJ - A REDOX-RESPONDING REPRESSOR OFBACTERIOCHLOROPHYLL, CAROTENOID, AND LIGHT HARVESTING-II GENE-EXPRESSION IN RHODOBACTER-CAPSULATUS, The Journal of biological chemistry, 272(29), 1997, pp. 18391-18396
Previous genetic analysis indicated that the photosynthesis gene clust
er from Rhodobacter capsulatus coded for the transcription factor, Crt
J, 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 an
d shown by gel mobility shift assays that CrtJ is biologically active.
DNase I footprint analysis confirms molecular genetic studies by show
ing that CrtJ binds to conserved palindromic sequences that overlap th
e -10 and -35 promoter regions of the bchC operon. Graphs of the perce
ntage 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) hi. Binding of CrtJ to the bchC promoter reg
ion was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-f
old higher binding affinity under oxidizing versus reducing conditions
.