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Nucleic Acids Research Advance Access published online on June 24, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp541
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© 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

Comparative analysis of activator-E{sigma}54 complexes formed with nucleotide-metal fluoride analogues

Patricia C. Burrows1, Nicolas Joly1, B. Tracy Nixon2 and Martin Buck1,*

1Department of Life Sciences, Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London, SW7 2AZ, UK and 2Department of Biochemistry and Molecular Biology, 406 Frear South Building, Pennsylvania State University, University Park, PA 16802, USA

*To whom correspondence should be addressed. Tel: +44 2075945442; Fax: +44 2075945419; Email: m.buck{at}imperial.ac.uk

Received May 6, 2009. Revised June 8, 2009. Accepted June 9, 2009.

Bacterial RNA polymerase (RNAP) containing the major variant {sigma}54 factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between {sigma}54-RNAP (E{sigma}54) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP–BeF- and ADP–AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the E{sigma}54 closed complex results in the re-organization of E{sigma}54 with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the E{sigma}54 closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex.


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