PMID:12484772
| Citation |
Tsujikawa, L, Strainic, MG, Watrob, H, Barkley, MD and DeHaseth, PL (2002) RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA. Biochemistry 41:15334-41 |
|---|---|
| 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 KMnO(4) 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. |
| Links | |
| Keywords |
2-Aminopurine/chemistry; DNA Footprinting; DNA, Bacterial/chemistry; DNA, Single-Stranded/chemistry; DNA-Directed RNA Polymerases/chemistry; Deoxyribonuclease I; Escherichia coli/enzymology; Escherichia coli/genetics; Fluorescence Polarization; Heparin/chemistry; Hot Temperature; Nucleic Acid Heteroduplexes/chemistry; Promoter Regions, Genetic; Spectrometry, Fluorescence; Templates, Genetic |
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