Intellectual merit: Genetic information is read by RNA polymerase II (pol II) through the synthesis of messenger RNA from DNA in the process termed transcription. A prerequisite for transcription is binding of transcription factor(s) to specific regulatory sequences on DNA, ultimately resulting in recruitment of pol II to a specific gene to initiate transcription. Since coordinate transcription by pol II defines networks of genes that function in the same biological process, elucidation of transcriptional regulatory mechanisms is essential to understanding biology. Mediator is an essential component that ensures correct temporal and spatial patterns of transcription by transducing the regulatory information through DNA-bound transcription activators to pol II. In the yeast Saccharomyces cerevisiae, Mediator is a large protein complex composed of 21 subunits with a total mass over 1 megaDalton. Despite its fundamental importance, the precise mechanism through which Mediator functions remains enigmatic. The current low-resolution picture has illuminated its modular organization into three distinct sub-complexes, termed Head, Middle and Tail respectively, but reveals no mechanistic insight. This project addresses a basic question, namely how this large multi-protein complex brings about proper transcription regulation. The working hypothesis to be tested is that the Mediator subunits provide key interacting domains for transcription factors and pol II, and that these specific interactions are essential for Mediator's function. The strategy will involve elucidation of the detailed structural features of Mediator combined with phenotypic and biochemical characterization of critical domains. Over the years, the size and complexity of Mediator has significantly hampered structural and functional studies of this kind. To overcome this problem, this project takes advantage of previous success in reconstituting one essential Mediator module, termed the Head (7 subunits with molecular mass of 223 kDa), in recombinant form. The Head module is essential for Mediator's function since it is required for promoter interaction in vivo and disabling it results in cession of mRNA synthesis. The goals of the project are (i) to solve the structure of the head module to an atomic resolution by X-ray crystallography, as well as (ii) to delineate the domain structure of the Head module subunits. Integration of the structural and functional insights will transform the current low-resolution information into a high-resolution structural and functional map of the Head module of Mediator. The map will illuminate how key regulatory domains are distributed within the complex, leading to delineation of distinct functional domains embedded within the structure of Mediator. Such structural and functional insight will transform the way Mediator is studied in the future.
Broader impacts: This project will provide an interdisciplinary research education that examines a basic mechanism of eukaryotic gene regulation by structural and functional studies of a multi-protein complex. The project will provide research training opportunities for Indiana University (IU) graduate students, as well as for undergraduate students from Indiana University Purdue University at Indianapolis (IUPUI) through the Life-Health Science Internship (LHSI) program. Students will gain experience with cutting-edge technologies including the latest molecular cloning and protein complex engineering technologies, X-ray crystallography, biochemical assays, and yeast genetics.
Intellectual Merit: The mapping of human genome is the unprecedented achievement of recent scientific history. After all, genes are ultimately the blueprint of every living organism including human beings. What is important, however, is to understand how cells use the information contained in the DNA to regulate almost every aspect of life such as growth and development and, eventually, aging. We have investigated the molecular machine called "Mediator", which players an essential role in reading the genetic information stored in DNA. Mediator is a gigantic molecular machine composed of 21 proteins. In this funding period, we successfully determined a structure of the key sub-complex of Mediator, termed the Mediator Head module, which is composed of 7 proteins (Figure 1A, 1B). The structure is remarkable, revealing how each protein interacts with one another to form highly integrated assemblies (Figure 1C). Notably, a portion of the Head named the "Neck domain"(Figure 2A) provides the stability and integrity of the complex by arranging the five proteins in a polymer-like structure, termed alpha-helical bundles (Figure 2B). An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits, and provides the binding sites for RNA polymerase II – an enzyme that transcribes DNA. Our results, in essence, reveal architectural principles underlying the role of Mediator in the regulation of gene expression. Moreover, the ability to solve such complex structures is important because multi-protein complexes such as Mediator will most likely become next generation of drug targets. Broader Impact: The project has provided the research opportunities for Indiana University (IU) graduate students, and the research training opportunity for the undergraduate students from Indiana University – Purdue University at Indianapolis (IUPUI) through the Life-Health Science Internship (LHSI) program. This project also provided the research opportunities for summer minority students through STEM scholar program as well as high school students through SEED (Summer Experienced for the Economically Disadvantaged) project. The students who participated our project had a chance to learn various cutting-edge technologies from molecular biology, complex protein complex expressions to X-ray crystallography. Significance and global impact of our work clearly been indicated by the coverage of our work: our structure work has been featured in NSF News from the Field; our structure was chosen as the "cover" of the Cold Spring Harbor Laboratory Meeting book for "Mechanisms of Eukaryotic Transcription"; our work has been featured in "Science & Research Highlights" at the Stanford Synchrotron Radiation Lightsource; and it was featured in Chem& Eng News. Finally, the results from our project is so fundamental that it certainly will be integrated into the lectures developed by the faculty members in other universities for both undergraduates and graduates in the future.
