All of the information required to generate a complete organism is stored in the DNA in discrete units called genes. To decode this information, a large enzyme complex known as RNA polymerase II is required. RNA polymerase II transcribes individual genes into mRNA molecules, which encode proteins (enzymes) that carry out chemical reactions in the cell or serve as structural frameworks to give cells and tissues their architecture. Not all genes in a given cell are transcribed at once, and determining how and when RNA polymerase II transcribes a given gene (there are ~30,000 in humans and 6,000 in yeast) is an area of intense study. Understanding this process, known as gene regulation, is the key to understanding how cells become different from one another during embryonic development, how cells respond to their environment, and how organisms evolve. Errors in gene regulation by RNA polymerase II contribute to disease and changes in fitness (survival) of an organism.
This project is aimed at understanding one level of control of the RNA polymerase II complex. RNA polymerase is composed of many protein subunits, the largest of which is called Rpb1. Rpb1 carries out the enzymatic action in which RNA is synthesized during transcription of a gene. Many smaller subunits interact with Rpb1 at different stages of transcription to help direct Rpb1 to the correct DNA site at the start of the gene (initiation), to help Rpb1 travel along the length of the gene (elongation), and to signal Rpb1 to stop transcription at the end of the gene (termination). Determining when these subunits bind to Rpb1 is an important process that appears to be aided by an enzyme called Ess1. Ess1 is known as a peptidyl-prolyl cis/trans isomerase. Ess1 binds to Rpb1 and is hypothesized to induce conformational changes (called cis/trans isomerizations) in Rpb1. In this way, Ess1 might control subunit binding to Rpb1, and thus help coordinate the multiple steps of transcription (initiation, elongation, termination) needed to generate mRNA and small RNAs. This research project is designed to determine exactly how Ess1 affects Rpb1, and thus how it may play a key role in gene regulation by the RNA polymerase II complex.
Broader impact of project: The research project will provide an opportunity for fulltime, intensive scientific training for a postdoctoral fellow and a Ph.D. student. Training will be in the various disciplines of genetics, biochemistry, and molecular biology. The project will also engage college undergraduate students during the summer months as part of the Department's Research Experience for Undergraduates Program (also sponsored by the National Science Foundation). Summer students will participate in ongoing experimental research during which time they will learn the scientific method, including the making of hypotheses, designing experiments to test them, carrying out the appropriate control experiments, as well as thoughtful analysis and interpretation of data. As such, the project should help inspire as well as train the next generation of biological researchers. In addition, the results of the project will be disseminated to the public in the form of research publications, presentations at national meetings, and free distribution of valuable reagents and scientific resources (data, strains, antibodies, etc.) to other qualified investigators.
This NSF-supported project was designed to help discover how information is decoded from DNA in all of our cells. An additional goal was to train young Ph.D. level scientists and to encourage college and high school students to become scientists by providing them with summer research opportunities. All goals were successfully met. Scientific progress: The genetic program that controls the structure and activity of all cells within an organism begins by decoding the information stored in the DNA of chromosomes. This information is first transcribed into RNA and then translated into proteins, the building blocks of the cell. In this project, we studied how the DNA template is converted into RNA, a processes known as "transcription." In particular, we studied regulation of an enzyme called RNA polymerase II, which carries out the transcription process in all animal cells. Regulation of RNA polymerase II is important because different cells must transcribe different genes to carry out their unique functions within the organism, for example heart cells that pump blood, brain cells that process external signals, blood cells that carry oxygen, etc. Our specific project was aimed at understanding how a small enzyme called Ess1 controls RNA polymerase II function. We used a model organism, Saccharomyces cerevisiae (baker’s yeast) because we could complete experiments much faster and better (and for less money) tha using human cells or mice. We found that Ess1 controls what helper proteins called "co-factors" will associate with RNA polymerase II. These co-factors help RNA polymerase II decide where to start and where to stop transcription of a given gene along the DNA. We found that Ess1 helps RNA polymerase II stop transcribing genes at their normal endpoints. Without Ess1, transcription proceeds into the next gene located further along the chromosome causing formation of abnormal RNAs that cannot be translated into proteins. The result is cell death. Our test-tube experiments show that Ess1 changes the shape of the RNA polymerase II enzyme, and we are interested in knowing exactly how that affects the interaction with co-factors within the cell. The details of this study are reported in two articles published in prominent scientific journals (listed below). These results were also presented to scientists from around the US and internationally at several universities and at scientific conferences. 1. Singh, N., Ma, Z., Gemmill, T., Wu, X., Rossettini, A., Rabeler, C., Beane, O., DeFiglio, H., Palumbo, M., Morse, R. and Hanes, S.D. (2009). The Ess1 prolyl isomerase is required for transcription termination of small non-coding regulatory RNAs via the Nrd1 pathway. Molecular Cell 36: 255-266. 2. Ma, Z., Atencio D., Barnes, C., DeFiglio, H., and Hanes, S.D. (2012) Multiple Roles for the Ess1 Prolyl Isomerase in the RNA Polymerase II Transcription Cycle. Molecular and Cellular Biology 32: 3594-3607. Broader Impacts - Training the next generation of research scientists: The research described above was carried out as part of the advanced training of two Ph.D. fellows (N. Singh and Z. Ma). Both completed their projects successfully and have gone on to other scientific positions. Both received extensive mentoring with respect to experimental science, scientific writing and speaking skills, and were able to present their work at international biology conferences. This NSF-supported project also enabled my laboratory to serve as a training ground for numerous high school and undergraduate students who have conducted summer research projects. This is an effective way to get students interested in science careers. Five college undergraduate students that worked in my laboratory have been co-authors on publications, and nearly all have entered Ph.D. doctoral programs. Thus, a key goal of this project has been met; to get young people interested in science and then serve as a springboard for their scientific careers. This NSF grant project has supported student research every year from 2006-2011. In addition, during this project, I also taught formal classes (in Molecular Genetics, and Developmental Biology) to doctoral students. I also served as a mentor for two junior faculty members, one at a historically black university (HBCU; Delaware State Univ.) and one here at SUNY-Upstate Medical University. In both cases, my role was to provide scientific and career advice (and help with grant-writing skills). In summary, student training & mentoring has been a large and we think successful part of this project.
