DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus.

Stephen N Ponnampalam,Carl E Bauer

doi:10.1074/jbc.272.29.18391

Stephen N Ponnampalam, Carl E Bauer

Open Access

https://doi.org/10.1074/jbc.272.29.18391

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Abstract

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.

Highlights

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
CrtJ Binding to the bchC Promoter Region Is Redox-sensitive—Since previous genetic analysis indicated that CrtJ most likely functions as an aerobic repressor [11], we addressed whether binding of CrtJ to the bchC promoter was affected by alterations in the redox state of the binding buffer
The isolated protein is active as evidenced from gel mobility shift assays where purified CrtJ shows an identical shift with that of the crude lysate of wild type R. capsulatus cells and by the results of our DNase I footprint titration assays

Summary

Introduction

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. 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. Genetic and transcriptional studies have indicated that crtJ appears to code for an aerobic repressor of bacteriochlorophyll (bch), carotenoid (crt), and light harvesting-II (puc) gene expression (8 –12). Gel mobility and DNase I protection analysis were utilized to demonstrate that CrtJ binds to a conserved palindrome sequence that is present in two copies of the bchC promoter. We demonstrate that binding of CrtJ to the bchC promoter is affected by the redox state of the binding buffer

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Conclusion