Nucleic Acids Research Advance Access published online on March 30, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp210
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Gene Regulation, Chromatin, and Epigenetics |
Transcription regulation of restriction-modification system Esp1396I
1Waksman Institute for Microbiology, Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854 USA, 2Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, 142292 Russia, 3Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth PO1 2DT, UK, 4E. A. Doisy Department of Biochemistry and Molecular Biology, St Louis University Medical School, St Louis, MO 63104, USA and 5Institutes of Molecular Genetics and Gene Biology, Russian Academy of Sciences, Moscow, Russia
*To whom correspondence should be addressed. Tel: +1 732 445 6095; Fax: +1 732 445 5735; Email: severik{at}waksman.rutgers.edu
Received January 27, 2009. Revised March 3, 2009. Accepted March 13, 2009.
The convergently transcribed restriction (R) and methylase (M) genes of the Restriction–Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vivo and in vitro. C-protein binding at the CR and M promoters was analyzed by DNA footprinting and a range of biophysical techniques. The distal and proximal C-protein binding sites at the CR promoter are responsible for activation and repression, respectively. In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter. Thus, during establishment of the system in a naïve host, the activity of the M promoter is turned off early, preventing excessive synthesis of methylase. Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought. Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.