RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA.
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.
Tsujikawa
Laura
L
Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Strainic
Michael G
MG
Watrob
Heather
H
Barkley
Mary D
MD
DeHaseth
Pieter L
PL
D015075:2-Aminopurine / Q000737:chemistry*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005454:Fluorescence Polarization; D006493:Heparin / Q000737:chemistry; D006358:Hot Temperature; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D011401:Promoter Regions, Genetic; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D015075:2-Aminopurine; D006493:Heparin; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I