RNA Polymerase Alters the Mobility of an A-Residue Crucial to Polymerase-Induced Melting of Promoter DNA

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Abstract
Strand separation in promoter DNA induced by Escherichia coli RNA polymerase is likely initiated at a conserved A residue at position −11 of the nontemplate strand. Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the −11 base. Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the −11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex. We demonstrate that the presence of 2AP instead of an A at position −11 has no major effect on the accessibility of DNA to DNase I or KMnO4 in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies. A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase. The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at −11 are immobilized in an RNA polymerase complex. This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparin-resistant complex with RNA polymerase. These results are consistent with the residue at −11 being tightly bound in a hydrophobic pocket of the enzyme.

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