Mechanism and Regulation of Early Trasncription by RNA polymerase II

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The expression of genes that are encoded by DNA is essential for sustained life. Transcription is the first step in gene expression. During transcription the enzyme RNA polymerase II, using DNA as a template, synthesizes mRNA molecules. The broad goal of this research is to understand in detail the steps that occur during early RNA polymerase II transcription and how they can be controlled. Such an understanding is important because transcription is a principal control point for regulating gene expression during growth, development, and normal cellular metabolism. Specifically, this research will identify which step in RNA synthesis is the slowest and therefore limits the rate at which mRNA is made. In addition, a factor in human nuclei that accelerates the rate of the slow step will be purified, identified, and characterized. This research will also investigate how large complexes containing proteins, DNA, and RNA, which are required for transcription to occur, both assemble and disassemble at genes that are being transcribed. The methods used in these studies center around a well established human transcription system that is assembled in vitro from highly purified proteins. The experiments utilize detailed quantitative assays specifically developed to study the steps of early RNA synthesis and their regulation. This will allow the identification of individual steps that dictate both the level and rate of RNA synthesis. In addition to the above research, an experimental module for an undergraduate biochemistry laboratory will be developed. This module will provide students with experience in designing and implementing a hypothesis driven experiment investigating protein-DNA interactions. Moreover, students will be exposed to state-of-the-art experimental techniques, thereby enhancing the scientific infrastructure of a teaching laboratory course. This research will impact the understanding of the critical first stages of mRNA transcription by defining where specific steps in the reaction occur and how they are interrelated, the role of the RNA transcript itself in early transcription, and how protein-DNA-RNA complexes assemble and disassemble. Moreover, the experiments will examine unique mechanisms by which transcription can be regulated. The knowledge derived from this research has the potential to stimulate new studies of transcriptional regulation in complex biological systems. In implementing this research, a significant amount of time will be spent training graduate students in experimental design, interpretation of data, creative thought, preparation and writing of manuscripts, and giving formal and informal oral presentations. In addition, developing an experimental module for an undergraduate laboratory underscores the importance of integrating research and education in order to promote teaching, training, and learning.