The long term objective of our work is to understand the molecular mechanisms underlying synergistic gene expression in eukaryotic cells. We will study how a well-characterized eukaryotic transactivator called ZEBRA differentially controls transcription of a family of target genes involved in the lytic cycle of Epstein-Barr virus. Our hypothesis is that the concentration of ZEBRA regulates the timing of target gene expression, and that different promoters respond to specific activator thresholds based on a promoter architecture that guides the cooperative assembly of upstream promoter-activator complexes and the ability of these complexes to recruit limiting components of the RNA polymerase II general transcription machinery to a gene. Our approach will be to correlate biochemical events in purified transcription systems with natural lytic expression patterns to establish a model for how a single activator can singularly control a genetic regulatory circuit.
Our specific aims are to i.) refine our knowledge of ZEBRA-mediated differential gene regulation in vivo and recreate expression of a subset of target genes in vitro; ii) employ purified ZEBRA, Sp-1 and HMG-1 in a study of how promoter architecture regulates assembly of nucleoprotein structures called enhanceosomes on target promoters; iii) investigate the biochemical mechanism by which activators recruit TFIIA, TFIID and TFIIB to a core promoter to nucleate assembly of a preinitiation complex and iv.) further study the biochemical details of a ZEBRA-mediated isomerization event in TFIID, necessary for full levels of gene activation in vitro. The mechanism by which a eukaryotic activator controls gene expression has represented one of the most intensively studied problems in contemporary biology, largely because of the central role aberrant gene expression plays in pathological processes. My laboratory has the technology to combine biochemical and biologic approaches towards understanding the molecular details of this central issue.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Biology Study Section (MBY)
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Tompkins, Laurie
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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Johnson, Kristina M; Wang, Jin; Smallwood, Andrea et al. (2004) The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly. Methods Enzymol 380:207-19
Johnson, Kristina M; Carey, Michael (2003) Assembly of a mediator/TFIID/TFIIA complex bypasses the need for an activator. Curr Biol 13:772-7
Johnson, Kristina M; Wang, Jin; Smallwood, Andrea et al. (2002) TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev 16:1852-63
Mitsouras, Katherine; Wong, Ben; Arayata, Charina et al. (2002) The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly. Mol Cell Biol 22:4390-401
Ellwood, K B; Yen, Y M; Johnson, R C et al. (2000) Mechanism for specificity by HMG-1 in enhanceosome assembly. Mol Cell Biol 20:4359-70
Ellwood, K; Huang, W; Johnson, R et al. (1999) Multiple layers of cooperativity regulate enhanceosome-responsive RNA polymerase II transcription complex assembly. Mol Cell Biol 19:2613-23
Wang, J; Ellwood, K; Lehman, A et al. (1999) A mathematical model for synergistic eukaryotic gene activation. J Mol Biol 286:315-25
Ellwood, K; Chi, T; Huang, W et al. (1998) Cooperative assembly of RNA polymerase II transcription complexes. Cold Spring Harb Symp Quant Biol 63:253-61