A direct real-time spectroscopic investigation of the mechanism of open complex formation by T7 RNA polymerase

Biochemistry
S S Sastry, B M Ross

Abstract

Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at...Continue Reading

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Citations

Feb 13, 2002·Biosensors & Bioelectronics·Christine N Jayarajah, Michael Thompson
Mar 21, 1998·Nucleic Acids Research·B HolzE Weinhold
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Apr 14, 2009·Methods : a Companion to Methods in Enzymology·Liang Zhao, Tianbing Xia
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Sep 8, 2010·Biochemistry·Thorben CordesAchillefs N Kapanidis

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