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Papers In Press, published online ahead of print September 15, 2005
Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, MO 63104
Corresponding Author: heydukt{at}slu.edu
We examined the relative affinity of model promoter constructs for binding E. coli RNA polymerase holoenzyme (RNAP). Model promoter constructs were designed to mimic DNA structures characteristic for different steps of transcription initiation. DNA duplexes in which a chemical crosslink was introduced just downstream from –10 hexamer to prevent DNA melting upon RNAP binding were used to mimic RNAP–promoter contacts in a closed complex. Fork junction DNA molecules with double-stranded/single-stranded junction between –11/–10 position were used to study interactions of RNA polymerase with DNA in open complex. The –35 and –10 promoter regions were found to be equally important for the initial RNAP binding. The recognition of –35 promoter region was independent of structural context of the model promoter fragment. In contrast, free energy of RNAP binding to –10 hexamer was highly dependent on DNA structure. The relative importance of –10 region for sequence-specific interaction with the polymerase was the lowest for constructs mimicking closed complex and the highest for the constructs mimicking open complex. The relative importance of region –10 was also dependent on the presence of –35 consensus element indicating a communication between different DNA binding determinants of polymerase during open complex formation. Short double-stranded promoter fragments comprising only –35 and –10 or only –10 consensus elements underwent melting in a complex with polymerase indicating that the core of promoter melting activity of the polymerase is localized to a very small subset of all promoter-polymerase contacts.
J. Biol. Chem, 10.1074/jbc.M507984200
Submitted on July 21, 2005
Revised on September 12, 2005
Accepted on September 15, 2005
E. coli RNA polymerase contacts outside the-10 promoter element are not essential for promoter melting
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