Nucleic Acids Research Advance Access originally published online on January 6, 2009
Nucleic Acids Research 2009 37(3):983-998; doi:10.1093/nar/gkn1010
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Nucleic Acids Research, 2009, Vol. 37, No. 3 983-998
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gene regulation, Chromatin and Epigenetics |
Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system
1Department of Medical Microbiology and Immunology, University of Toledo Health Sciences Campus, Toledo, OH 43614-2598, USA, 2Department of Microbiology, University of Gdansk, Gdansk, 80-822, Poland and 3Program in Bioinformatics and Proteomics/Genomics, University of Toledo Health Sciences Campus, Toledo, OH 43614-2598, USA
*To whom correspondence should be addressed. Tel: +1 419 383 5422; Fax: +1 419 383 3002; Email: Robert.Blumenthal{at}utoledo.edu
Received October 24, 2008. Revised November 26, 2008. Accepted December 3, 2008.
Most type II restriction-modification (R-M) systems produce separate endonuclease (REase) and methyltransferase (MTase) proteins. After R-M genes enter a new cell, MTase activity must appear before REase or the host chromosome will be cleaved. Temporal control of these genes thus has life-or-death consequences. PvuII and some other R-M systems delay endonuclease expression by cotranscribing the REase gene with the upstream gene for an autogenous activator/repressor (C protein). C.PvuII was previously shown to have low levels early, but positive feedback later boosts transcription of the C and REase genes. The MTase is expressed without delay, and protects the host DNA. C.PvuII binds to two sites upstream of its gene: OL, associated with activation, and OR, associated with repression. Even when symmetry elements of each operator are made identical, C.PvuII binds preferentially to OL. In this study, the intra-operator spacers are shown to modulate relative C.PvuII affinity. In light of a recently reported C.Esp1396I-DNA co-crystal structure, in vitro and in vivo effects of altering OL and OR spacers were determined. The results suggest that the GACTnnnAGTC consensus is the primary determinant of C.PvuII binding affinity, with intra-operator spacers playing a fine-tuning role that affects mobility of this R-M system.