Intellectual Merit: RNA polymerase II (Pol II) reads genetic information by synthesizing messenger RNA from DNA in the process termed transcription. Mediator is a large multi-protein complex that regulates most, if not all, gene transcription by Pol II. It functions as a co-activator by conveying signals from DNA regulatory sequences to Pol II and a set of general transcription factors (GTFs) that assemble at the promoter to form a pre-initiation complex (PIC) for transcription initiation. Mediator is evolutionarily conserved in all eukaryotes. In the yeast Saccharomyces cerevisiae, Mediator is composed of 21 subunits, with a total mass of over 1 megaDalton, organized into three three distinct modules, termed Head, Middle/Arm and Tail. Since Mediator's function in PIC assembly is proposed to involve direct interactions with Pol II and GTFs, structural information of functional surfaces within Mediator that interact with GTFs and Pol II is required to understand the mechanisms of Mediator functions. However, the large size and complexity of Mediator has greatly impeded structural studies of these questions. This fundamental bottleneck has been overcome recently by determining a crystal structure of the Mediator Head module at 4.3A resolution. This Mediator module comprises 7 subunits with a molecular mass of 223 kDa and is essential for Mediator function. The Head structure revealed a number of structural features that may be responsible for recruitment and coordination of the PIC. These observations led to the hypothesis that Mediator provides functional surfaces with which transcription factors and Pol II interact to bring about transcriptional regulation. To test this hypothesis, this project will determine functional surfaces by taking a structure-guided mutagenesis approach to improve the resolution of the current structure. Structural and functional studies will be combined to determine how the Head module of Mediator interacts with a module of Pol II. Together, the results will provide a picture of how Mediator spatially coordinates different components of the transcription machinery to facilitate a sequence of event leading to transcription initiation. Such structural and functional insights will greatly transform understanding of gene expression regulation by Mediator.
Broader Impacts: This project will provide research training opportunities for Indiana University graduate students and for undergraduate students from Indiana University, Purdue University at Indianapolis through the Life-Health Science Internship program. Research training opportunities will also be available for summer students through the STEM program for undergraduate minority students and the Indy SEED project for underprivileged high school students. Students will learn and implement cutting-edge technologies, including the latest molecular cloning and protein complex engineering technologies, X-ray crystallography, bioinformatics, biochemical assays, and yeast genetics.
