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James A. GOODRICH Office Phone: 303 492 3273
E-mail: James.Goodrich@colorado.edu
FAX: 303 492 5894
Lab: Cristol Chemistry 218, 220
Lab Phone: 303 492 2508
Group Website: www.colorado.edu/chemistry/goodrichlab/
Ph.D.: Carnegie Mellon University, 1992
Postdoctoral Fellow: University of California at Berkeley, 1992-96
Damon Runyon-Walter Winchell Cancer Fund, 1992-94
MAMMALIAN mRNA TRANSCRIPTION AND ITS REGULATION
Controlling gene expression is essential to growth, development, and sustained life. A critical control point for regulating gene expression is at the level of transcription. The proper regulation of transcription is essential for maintaining normal pathways of cell growth and differentiation, thereby avoiding the rampant cell proliferation observed in tumors. Transcription of protein encoding genes in eukaryotes is orchestrated by a host of protein factors, including RNA polymerase II, general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), coactivators, chromatin remodeling factors, and gene-specific transcriptional regulators (activators and repressors). The underlying goal of our work is to uncover molecular mechanisms governing mammalian RNA polymerase II transcription. To this end, we use a combination of biochemistry, molecular biology, and molecular genetics to investigate mechanisms of transcriptional regulation in three lines model systems.
Regulation of RNA Polymerase II by Small RNAs
All cells respond to stress, and do so in part by altering gene expression. When eukaryotic cells are subjected to heat shock, general RNA polymerase II transcription decreases at the same time as transcription of a set of heat shock specific genes increases. While the activation of heat shock specific genes has been studied in great detail, very little is understood about how general mRNA transcription is repressed in response to environmental stresses like heat shock. A small non-translated mouse RNA (B2 RNA) is transcriptionally upregulated upon heat shock. We have found that B2 RNA binds RNA polymerase II with high affinity (low nM) and blocks the formation of functional initiation complexes in vitro. These findings provide a model for the mechanism of transcriptional repression during heat shock. We are currently investigating the role of other small RNAs in regulating mouse and human transcription.
Regulation of Transcription at the Human Interleukin-2 Gene
The mammalian immune system represents a unique model for studying the importance of transcriptional regulation in governing cell growth and differentiation. Studies performed by immunologists have revealed that the development of T-lymphocytes is controlled by the interplay of signal transduction and transcription. Interleukin-2 is a cytokine that acts as an autocrine growth factor promoting the proliferation and development of T cells during the immune response to bacterial and viral infection, as well as tumorigenesis. The IL-2 promoter is relatively compact for mammalian genes, since proper regulation of IL-2 transcription requires only 340 bp of DNA surrounding the transcription start site. Although small, the IL-2 promoter has a complex regulatory region that contains binding sites for at least four families of transcriptional regulatory proteins: NFAT, NF-kB, AP1 (cJun and cFos), and OCT.
We are investigating the roles of both cis-regulatory elements and trans-regulatory factors (e.g., NFAT, AP1, and OCT) in transcription at the IL-2 promoter using in vitro transcription experiments and assays in T cells. Protein-protein contacts are important for transcriptional regulation. For example, contacts between a transcriptional activator and components of the transcription machinery often represent an early step in the process of transcriptional activation. We are performing a series of protein-protein interaction assays to identify and characterize the molecular targets of NFAT, cJun, and cFos in the basal transcription machinery. Our studies revealed that the activators NFATp and cJun target coactivator subunits of the TFIID complex. Human TAFII130 (hsTAF4) is a coactivator for NFATp while cJun derepresses transcription by binding the N-terminal region of TAFII250 (hsTAF1). The functions of these interactions are being investigated through a combination of mutagenesis and transcription assays. The in vitro experiments are being complemented with cell-based experiments to test the effects of specific protein-protein interactions on the development of the mammalian immune response. The effect of chromatin on IL-2 transcription is also being studied.
The mechanism of the human RNA polymerase II transcription reaction
The goal of this line of research is to understand what limits the rate and extent of transcription at human promoters. We reconstituted human RNA polymerase II transcription in vitro using highly purified transcription factors and used this system to measure the kinetics of discrete steps in the transcription reaction: including, preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation. Our studies revealed that promoter escape can limit the rate of transcription. The finding that promoter escape is rate limiting in vitro suggests that this may be an important step for regulation at natural promoters in the human genome. In performing kinetic studies we identified a transition, termed escape commitment, which occurs after initiation and prior to promoter escape. Upon completion of escape commitment, ternary complexes are stable and slowly proceed forward through promoter escape. Escape commitment involves translocation between synthesis of the third and fourth phosphodiester bonds. We propose that a conformational change in ternary transcription complexes occurs during translocation after synthesis of a 4 nucleotide RNA. Ongoing studies are investigating how the promoter DNA, transcript RNA, and general transcription factors contribute to identified and yet uncharacterized transformations that occur during early RNA synthesis.
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.
Selected Publications
Weaver, J.R., Good, K., Walters, R.D., Kugel, J.F., and Goodrich, J.A. (2007). Characterization of the sequence and architectural constraints of the regulatory and core regions of the human interleukin-2 promoter. Mol. Immunol. 44: 2813-2819.
Espinoza, C.A., Goodrich, J.A., and Kugel, J.F. (2007). Characterization of the structure, function and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription. RNA. 13: 583-596.
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.
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. Nature Struct. Mol. Biol. 11: 822-829.
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. Nature Struct. Mol. Biol. 11: 816-821.
Lively, T.N., Nguyen, T.N., Galasinski, S.K., and Goodrich, J.A. (2004). The basic leucine zipper domain of cJun functions in transcriptional activation through interaction with the N‑terminus of hsTAF1 (human TAFII250). J. Biol. Chem. 279: 26257-26265.
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.
Ferguson, H.A. and Goodrich, J.A. (2001). Expression and purification of recombinant human c‑Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Nucleic Acids Res. 29: E98 (6 pages).
Lively, T.N., Ferguson, H.A., Galasinski, S.K., Seto, A.G., and Goodrich, J.A. (2001). c‑Jun binds the N‑terminus of human TAFII250 to derepress RNA polymerase II transcription in vitro. J. Biol. Chem. 276: 25582-25588.
Kim, L.J., Seto, A.G., Nguyen, T.N., and Goodrich, J.A. (2001). Human TAFII130 is a coactivator for NFATp. Mol. Cell. Biol. 21: 3503-3513.
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.
Kim, L.J., Ferguson, H.A., Seto, A.G., and Goodrich, J.A. (2000). Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp. BMC Immunology 1: 1 (10 pages).
Galasinski, S.K., Lively, T.N., Grebe de Barron, A., and Goodrich, J.A. (2000). Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones. Mol. Cell. Biol. 20: 1923-1930.
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.
Reviews, Methods, Chapters, News and Views
Kugel, J.F. and Goodrich, J.A. (2007). An RNA transcriptional regulator templates its own regulatory RNA. Nature Chem. Biol. 3: 89-90.
Goodrich, J.A. and Kugel, J.F. (2006). Noncoding RNA regulators of RNA polymerase II transcription. Nature Rev. Mol. Cell Biol. 7: 612-616.
Kugel, J.F. and Goodrich, J.A. (2006). Beating the heat: a translation factor and an RNA mobilize the heat shock transcription factor HSF1. Mol. Cell. 22: 153-154.
Nguyen, T.N. and Goodrich, J.A. (2006). Immobilized protein-protein interaction assays: Eliminating false positive interactions caused by contaminating nucleic acid. Nature Methods. 3: 135-139.
Goodrich, J.A. and Tjian, R. (2006). Transcription: The never ending story. In Gene Expression and Regulation. Ma, J., ed. (Beijing: Higher Education Press and Springer).
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.
Books
Goodrich, J.A. and Kugel, J.F. (2006). Binding and Kinetics for Molecular Biologists. Cold Spring Harbor Laboratory Press. 182 pages. Computer simulations: http://kinetics.cshl.edu/.
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