Abstract
The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm. This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12. The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1). Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1).
Highlights
The bacteriophage RNA polymerases, such as the T7 RNA polymerase, are single subunit enzymes capable of catalyzing all the processes such as initiation, elongation, and termination of transcription
All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner
Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at ؊4 from the guanine at ؊5, during open complex formation
Summary
The bacteriophage RNA polymerases, such as the T7 RNA polymerase, are single subunit enzymes capable of catalyzing all the processes such as initiation, elongation, and termination of transcription. In the absence of the initiating nucleotide, GTP, T7 RNA polymerase melts Ϫ4 to ϩ2 region, which includes the TATA sequence, and in the presence of GTP, the unpaired region extends from Ϫ4 to ϩ4 [4] Our goal in these studies was to determine the kinetic and thermodynamic parameters that govern the formation of the closed and open complexes. We have systematically substituted each of the adenines in the Ϫ4 to ϩ4 region with 2-AP, and measured the kinetics of the fast form of the T7 RNA polymerase with these DNAs. The fluorescence stopped-flow studies have provided important structural insights and a more detailed multistep mechanism of the initiation process, including the rate constant and equilibrium constant of promoter opening and the associated conformational changes
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