A Direct Real-Time Spectroscopic Investigation of the Mechanism of Open Complex Formation by T7 RNA Polymerase

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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 −4 was also slower in the presence of GTP than in its absence. At 37 °C, base pair disruption occurred first at −1 followed by −6 and finally at −4. Open complex formation was temperature-sensitive. Temperature effects at −1, −6, and at −4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around −1 and −6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at −1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near −1 and −6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at −1 and at −6, which are followed by bidirectional propagation to −4.