Regulation of transcription by RNA polymerase II
Regulating the levels at which genes are expressed is central to all of biology. A primary point for controlling gene expression is during mRNA transcription, which occurs as the enzyme RNA polymerase II synthesizes an RNA copy of the DNA in protein encoding genes. The ultimate goal of our research is to arrive at a detailed understanding of the RNA polymerase II transcription reaction and how it is regulated. There are three broad lines of research in the lab.
Mechanism of the 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.
Small RNA regulators of RNA polymerase II
In response to heat shock, cells execute a program of gene-specific transcriptional activation and repression. We have found that during the heat shock response in mouse cells, a small non-coding RNA polymerase III transcript, B2 RNA, associates with RNA polymerase II and represses transcription of specific mRNA genes. It does so by binding to core RNA polymerase II with high affinity and specificity. We have shown in vitro that B2 RNA assembles into preinitiation complexes at promoter DNA and blocks all detectable RNA synthesis. These studies define a unique transcriptional regulatory mechanism involving an RNA regulator. Moreover, they identify a function for B2 RNA, which is transcribed from short interspersed elements (SINEs) that are abundant in the mouse genome and historically considered to be 'junk DNA'. Because we are ultimately interested in understanding the regulation of human transcription, we are also identifying and studying human RNA(s) that control RNA polymerase II transcription.
Regulating transcription of the human interleukin-2 gene
The cytokine interleukin-2 (IL-2) is a key signaling molecule that controls the mammalian immune response to infection. Proper regulation of IL-2 expression is critical for clonal proliferation and differentiation of T cells after stimulation with foreign antigen. A critical control point for regulating levels of IL-2 is during transcription. Production of IL-2 in response to T cell stimulation is regulated by at least four families of transcriptional activators: NFAT, NF-kB, OCT, and AP1 (cJun and cFos). We have identified a series of protein-protein interactions in vitro that occur between activators that bind at the IL-2 promoter as well as between activators and coactivators. Specifically, these include interactions between cJun and TAF1, NFAT1 and TAF4, and cJun and NFAT1 (independent of DNA). We have also characterized how promoter elements contribute to setting the level of transcription in cells and the rate of transcription in vitro by using an extensive series of mutations in the regulatory and core regions of the IL-2 promoter. The goal of future research is to understand how specific protein-protein interactions, promoter sequence, and transcriptional regulators work together to set levels of IL-2 transcription in cells. Ultimately, we would like to determine whether the protein-protein interactions we identified are important for transcriptional activation at genes other than IL-2.
Goodrich, J.A. and Kugel, J.F. (2006) Non-coding-RNA regulators of RNA polymerase II transcription. Nat. Rev Mol. Cell Biol. In press.
Heib, A., 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. In press.
Weaver, J.R., Kugel, J.F., Goodrich, J.A. (2005) The sequence at specific positions in the early transcribed regions sets the rate of transcript synthesis by RNA polymerase II in vitro. J. Biol. Chem. 48: 39860-39869.
Allen, T.A., Von Kaenel, S., Goodrich, J.A., and Kugel, J.F. (2004) The SINE encoded mouse B2 RNA represses mRNA transcription in response to heat shock. Nat. Struct. Mol. Biol. 11: 816-821.
Espinoza, C.A., Allen, T.A., Hieb, A. R., Kugel, J.F., and Goodrich, J.A. (2004) B2 RNA binds directly to RNA polymerase II to repress transcript synthesis. Nat. Struct. Mol. Biol. 11: 822-829.
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.