Nucleic Acids Research Advance Access originally published online on August 19, 2009
Nucleic Acids Research 2009 37(18):5981-5992; doi:10.1093/nar/gkp658
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Nucleic Acids Research, 2009, Vol. 37, No. 18 5981-5992
© The Author 2009. Published by Oxford University Press.
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 |
The role of the conserved phenylalanine in the 
54-interacting GAFTGA motif of bacterial enhancer binding proteins
1Division of Biology, Sir Alexander Fleming Building and 2Department of Microbiology, Division of Investigative Sciences, Faculty of Medicine and Centre for Molecular Microbiology and Infection, Flowers Building, Imperial College London, London SW7 2AZ, UK
*To whom correspondence should be addressed. Tel: +44 2075945366; Fax: +44 2075945419; Email: n.joly{at}imperial.ac.uk
Correspondence may also be addressed to Martin Buck. Tel: +44 2075945442; Fax: +44 2075945419; Email: m.buck{at}imperial.ac.uk
Received June 12, 2009. Revised July 22, 2009. Accepted July 23, 2009.
54-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns. Here we use Escherichia coli PspF, a model bEBP involved in the transcription of stress response genes (psp operon), to study determinants of its contact features with the closed promoter complex. We demonstrate that substitution of a highly conserved phenylalanine (F85) residue within the L1 loop GAFTGA motif affects (i) the ATP hydrolysis rate of PspF, demonstrating the link between L1 and the nucleotide binding pocket; (ii) the internal organization of the hexameric ring; and (iii) 54 interactions. Importantly, we provide evidence for a close relationship between F85 and the –12 DNA fork junction structure, which may contribute to key interactions during the energy coupling step and the subsequent remodelling of the E54 closed complex. The functionality of F85 is distinct from that of other GAFTGA residues, especially T86 where in contrast to F85 a clean uncoupling phenotype is observed.