Peculiar 2-aminopurine fluorescence monitors the dynamics of open complex formation by bacteriophage T7 RNA polymerase.
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).
Bandwar
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Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Patel
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D015075:2-Aminopurine / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014764:Viral Proteins
D014764:Viral Proteins; D015075:2-Aminopurine; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases