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J. Biol. Chem., Vol. 278, Issue 52, 52944-52952, December 26, 2003

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Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein^*

David J. Lee, Stephen J. W. Busby, and Georgina S. Lloyd

From the School of Biosciences, the University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

The C-terminal domain of the subunit (CTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that CTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of CTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase subunit. After incorporation of the tagged subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of CTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.

Received for publication, July 30, 2003 , and in revised form, September 24, 2003.

^* This work was supported by project grants from the UK Biotechnology and Biological Sciences Research Council and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-121-414-5435; Fax: 44-121-414-7366; E-mail: G.S.Lloyd{at}bham.ac.uk.

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