Intellectual Merit:Genetic information is read by RNA polymerase II (Pol II) by synthesizing messenger RNA from DNA in the process termed transcription. Since transcription by Pol II defines biological activities in eukaryotes, elucidation of transcription process is essential to understand biology. Mediator is an essential regulator in transcription. In the yeast Saccharomyces cerevisiae, it is a large protein complex composed of 25 subunits with a total mass over 1 mega Daltons. Mediator is organized into 4 distinct sub-complexes termed, Head, Middle, Tail and the CDK8 modules. Head, Middle and Tail modules combined form a very stable so-call core Mediator (21-subunits); the CDK8 module (4 subunits) is dissociable from core Mediator and in some cases, it may function by its own. Despite of its fundamental importance, the mechanism of its function is largely unknown. To elucidate the mechanism, structure and functional characterization of the Mediator complex is essential. Our investigation has led to two major outcomes as follows:
(i) Elucidation of the mechanism of transcription by Mediator: our investigation has revealed key regulatory mechanism of Mediator in transcription: the key role of Mediator is to recruit the one of the general transcription factors (GFTs), TFIIH, which will in turn facilitate transition of Pol II from initiation to elongation. While core Mediator facilitates transition of Pol II from initiation to elongation, the CDK8 module inhibits recruitment of TFIIH. In essence, we found that Mediator functions to regulate the efficiency of transition of Pol II from initiation to elongation by facilitation or inhibition of recruitment of TFIIH (Figure 1).
(ii) Development of the Solubility-Enhancing-Protein assisted protein expression (SEP) system and its application to reconstitution of the entire Mediator: The biggest obstacle to reconstituting large multi-protein complexes lies in a difficulty expressing large subunits in soluble form. To solve this problem, we developed a unique baculovirus expression vector that allows for the expression of large proteins in insect cells. This unique vector system fuses an engineered polypeptide to the protein of interest, drastically improving the expression, solubility and stability of numerous otherwise problematic large proteins. We have named this technology “Solubility-Enhancing-Protein assisted protein expression (SEP) system” (unpublished, a provisional patent application has been filed). Using our new technology, we have succeeded reconstitution of the 18- subunit as well as the 21-subunit Mediator (Figure 2A, 2C). The preliminary EM data is shown in Figure 3D. We also succeeded in reconstituting the CDK8 module (Figure 2E). We obtained the low-resolution 3D reconstitution of the CDK8 module (Figure 3F). These outcomes will lead to structure determination of the entire 25-subunit Mediator complexes. Moreover, We believe that our SEP technology will become an important protein-engineering tool in the future.
Our investigation has produced: 5 research papers, 2 review papers, one Japanese textbook chapter, one manuscript in revision, and 3 manuscripts are in preparation. In addition, one provisional patent application has been filled.
Broader impacts: This project has provided the research training opportunities for the undergraduate students from Indiana University – Purdue University at Indianapolis (IUPUI) through the Life-Health Science Internship (LHSI) program: a total of 5 students participated during the funding period. This project also provides the research training opportunities to summer students from the STEM program (undergraduate minority students from Jackson State University) as well as the SEED project for underprivileged high school students. In our training programs, students have experienced cutting-edge technologies including the latest molecular cloning and protein complex engineering technologies, X-ray crystallography, and yeast genetics.
The main outcome of our educational outreach efforts is that a majority of former students, mostly either minority or underprivileged, have gone to pursue career in STEM fields as follows:
One female former student from LHSI program went to University of Notre Dame to obtain a master’s degree and is currently working in a pharmaceutical company (Eli Lilly). Another former female student from LHSI program is working at Volkswagen America as an engineer. 3 of 5 minority female students are currently in PhD program (University of Michigan, UC Santa Barbara, Old Dominion University). 2 students in SEED projects have gone to Indiana University (IU) as well as IUPUI: one minority student already graduated and other former female student is in her senior year, applying for a medical school. Again, a majority of former students have gone to pursue career in STEM fields, showcasing my laboratory’s commitments to broader impacts.
Overall, our research project aimed to understand the structure and functions of Molecular Machine. Visualizing a large molecular machine involved in fundamental cellular process in life will undoubtedly captivate imaginations of many people, underscoring a true value of basic science.
Last Modified: 08/01/2017
Modified by: YuichiroTakagi
