The goal of this proposal is to explore the structure and function of the RNA polymerase II transcription initiation complex using a combination of genetic and biochemical assays. The yeast Saccharomyces cerevisiae provides an ideal system for carrying out coordinated in vivo and in vitro studies that are not feasible in other model systems. The yeast transcription machinery is highly homologous to that in mammalian cells, so findings in one system can be extended to the other. The studies proposed here will contribute to our understanding of gene expression and of how defects in this process lead to cancer or developmental abnormalities. In addition, understanding the specific details of transcription in yeast may provide useful information for designing antifungal drugs. Genes for specific basal transcription factors will be mutagenized and introduced into yeast cells. Mutant genes that confer interesting phenotypes (e.g. high or low temperature sensitivity, inability to grow on certain media) will be further characterized. Mutants will be analyzed in the genes encoding the TATA Binding Protein (TBP), the TAF subunits of TFIID, TFIIB, the Kin28 subunit of TFIIH, and both subunits of TFIIE. In addition, two new bromodomain-containing proteins that interact with TFIID have been isolated and will be characterized for their role in transcription. Many of the basal transcription factors interact with each other, so biochemical assays such as in vitro transcription, native gel electrophoresis, immobilized template assay, and co- immunoprecipitation will be used to assay protein-protein interactions with the mutant proteins. Defects in specific biochemical functions will be correlated with mutations in specific residues or regions of the transcription factors. It is expected that this approach will allow specific activities and protein-protein contacts to be studied. In cases where mutations in one transcription factor disrupt interactions with a second factor and thereby cause a conditional phenotype, suppressors in the second factor gene will be isolated that restore growth at the restrictive temperature. Again, the in vivo behavior of the mutant-suppressor pairs will be explored by testing protein-protein interactions in vitro. Also, random mutagenesis will be used to isolate second-site suppressors of mutant transcription factors. The proteins identified as suppressors (whether known factors or new proteins) will be characterized as to their interactions with the transcription machinery.
| Joo, Yoo Jin; Ficarro, Scott B; Soares, Luis M et al. (2017) Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation. Genes Dev 31:2162-2174 |
| Woo, Hyeonju; Dam Ha, So; Lee, Sung Bae et al. (2017) Modulation of gene expression dynamics by co-transcriptional histone methylations. Exp Mol Med 49:e326 |
| Soares, Luis M; He, P Cody; Chun, Yujin et al. (2017) Determinants of Histone H3K4 Methylation Patterns. Mol Cell 68:773-785.e6 |
| Kim, Ji Hyun; Lee, Bo Bae; Oh, Young Mi et al. (2016) Modulation of mRNA and lncRNA expression dynamics by the Set2-Rpd3S pathway. Nat Commun 7:13534 |
| Hahn, Steven; Buratowski, Stephen (2016) Structural biology: Snapshots of transcription initiation. Nature 533:331-2 |
| Soares, Luis M; Radman-Livaja, Marta; Lin, Sherry G et al. (2014) Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns. Cell Rep 6:961-972 |
| Marquardt, Sebastian; Escalante-Chong, Renan; Pho, Nam et al. (2014) A chromatin-based mechanism for limiting divergent noncoding transcription. Cell 157:1712-23 |
| Suh, Hyunsuk; Hazelbaker, Dane Z; Soares, Luis M et al. (2013) The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits. Mol Cell 51:850-8 |
| Soares, Luis M; Buratowski, Stephen (2013) Histone Crosstalk: H2Bub and H3K4 Methylation. Mol Cell 49:1019-20 |
| Weiner, Assaf; Chen, Hsiuyi V; Liu, Chih Long et al. (2012) Systematic dissection of roles for chromatin regulators in a yeast stress response. PLoS Biol 10:e1001369 |
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