The mechanism of the human RNA polymerase II transcription reaction
The mechanism of the human RNA polymerase II transcription reaction A primary goal of our research is to understand the mechanism and regulation of early transcription. The foundation of our studies is a human in vitro transcription system that is reconstituted from highly purified general transcription factors and RNA polymerase II. Using this system, we have developed techniques to experimentally dissect the transcription reaction into 5 steps: preinitiation complex formation, initiation, escape commitment, promoter escape, and elongation. We have arrived at a kinetic model for a single round of transcription by measuring forward and reverse rate constants for each of the aforementioned steps. In addition, we have identified the precise position of the rate-limiting step, which completes promoter escape and demarcates the formation of an elongation complex. Our kinetic model provides the framework for future studies aimed at investigating new transformations during early transcription, and determining how regulatory factors affect the kinetics of specific steps in the reaction.
A working model for the RNA polymerase II transcription reaction. Abbreviations: R, RNA polymerase II and the general transcription factors; P, promoter DNA; PIC, preinitiation complex; RPI, initiated complex; RPEC, escape committed complex; and RE, elongation complex.
Publications
Hieb, A.R., Baran, S., Goodrich, J.A., and Kugel, J.F. (2006). An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation. EMBO J. 25: 3100-3109.
Weaver, J.R., Kugel, J.F., and Goodrich, J.A. (2005). The sequence at specific positions in the early transcribed region sets the rate of transcript synthesis by RNA polymerase II in vitro. J. Biol. Chem. 280: 39860-39869.
Kugel, J.F. and Goodrich, J.A. (2003). In vitro studies of the early steps of RNA synthesis by human RNA polymerase II. Methods Enzymol. 370: 687-701.
Kugel, J.F. and Goodrich, J.A. (2002). Translocation after synthesis of a four nucleotide RNA commits RNA polymerase II to promoter escape. Mol. Cell. Biol. 22: 762-773.
Ferguson, H.A., Kugel, J.F., and Goodrich, J.A. (2001). Kinetic and mechanistic analysis of the RNA polymerase II transcription reaction at the human interleukin-2 promoter. J. Mol. Biol. 314: 993-1006.
Kugel, J.F. and Goodrich, J.A. (2000). A kinetic model for the early steps of RNA synthesis by human RNA polymerase II. J. Biol. Chem. 275: 40483-40491.
Kugel, J.F. and Goodrich, J.A. (1998). Promoter escape limits the rate of transcription from the adenovirus major late promoter on negatively supercoiled templates. Proc. Natl. Acad. Sci. 95: 9232-9237.