Abstract
We have used stopped-flow and rapid chemical quench-flow methods to investigate the kinetics of the early steps during transcription initiation by bacteriophage T7 RNA polymerase. Most promoters of T7 RNA polymerase initiate with two GTPs. The kinetics of GTP binding was investigated by monitoring the fluorescence changes resulting from GTP binding to polymerase and fluorescent 2-aminopurine-containing promoter DNA complex. Scheme 1 was determined from studies of T7 Phi10 promoter at 25 degrees C, where (E.D)n represents the polymerase.DNA complex in different conformations. GTPE and GTPI represent the elongating and initiating GTP molecules incorporated at the +2 and +1 positions, respectively. Our studies show that GTP at the elongation site binds with at least 10-fold tighter affinity than the GTP at the initiation site. Two conformational changes were revealed upon GTP binding to the polymerase.2-aminopurine DNA complex. The first conformational change occurred upon GTP binding to the elongation site. This conformational change was reversible, and studies with partially melted DNA and incorrect NTPs suggested that it may represent a DNA melting and/or base pairing step. A second rate-limiting conformational change whose rate was same as the maximum rate of pppGpG synthesis occurred after two GTPs were bound. As with DNA polymerases, this rate-limiting conformational change probably occurs at each NMP incorporation event and may be involved in proper positioning of the initiation and the elongating GTPs within the polymerase active site to achieve efficient and accurate RNA synthesis.
Highlights
Of the kinetics and thermodynamics of the promoter binding during transcription initiation has been a subject of numerous investigations [1, 2], very little is known about the details of the steps following promoter binding
Whereas the interactions between the polymerase and the promoter DNA have been studied to some extent, very little is known about the kinetic mechanism of initiation and promoter clearance
Using presteady-state kinetics we have shown that T7 RNA polymerase binds to the promoter DNA with diffusion-limited rate constants, and promoter opening is a fast step relative to the rate of RNA synthesis during initiation [1, 5]
Summary
T7 RNA Polymerase Purification—T7 RNA polymerase was purified as described before [5]. Three chromatographic columns consisting of SP-Sephadex, CM-Sephadex, and DEAE-Sephacel (all purchased from Sigma Chemical Co.) were used to purify the RNA polymerase from Escherichia coli strain BL21/pAR1219 [10]. The polymerase was Ͼ95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. The purified enzyme was stored in buffer (20 mM sodium phosphate, pH 7.7, 1 mM Na3-EDTA, and 1 mM dithiothreitol) containing 100 mM NaCl and 50% (v/v) glycerol at Ϫ80 °C.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
