Gene regulation is a fundamental process in cells and alterations in gene expression have been linked to numerous disease states in humans. Genes are regulated at multiple levels and determining how these events are coordinated is critical to understanding cellular physiology. The central enzyme in this process is RNA Polymerase II (RNAPII), which syntheses mRNAs. RNAPII encounters many types of barriers during transcription, which are overcome by protein complexes called elongation factors (EFs). Two formable roadblocks for RNAPII are the nucleosome and DNA damage, which slows or prevents the completion of mRNAs across genes. The goal of this proposal is to understand how RNAPII contends with these two barriers to transcription. We have identified two conserved EFs, Spt4/5 (DSIF in metazoans) and Ccr4-Not as playing an important role in allowing RNAPII to contend with these barriers. We have a developed approaches that fully exploit the biochemistry, powerful genetics and molecular biology of yeast to characterize the mechanism of elongation control. Spt4/5 and Ccr4-Not are conserved across the eukaryotic kingdom; thus, yeast is an appropriate model to study the function of these two EFs. The first goal of the studies described in this proposal is to investigate how the Spt4/5 complex aids RNAPII passage through the nucleosome and identify the domains of its large subunit, Spt5, required for this function. The second goal is to characterize the role of the Ccr4-Not complex in regulating the transcription of RNAPII through damaged DNA templates. We will determine if Ccr4-Not directly modifies the large subunit of RNAPII, Rpb1, and/or serves as a scaffold to recruit factors that remove RNAPII from sites of DNA damage. Third, we use an unbiased mass spectrometry (MS) proteomics screen based on stable isotope labeling by amino acids in culture (SILAC) to identify and verify novel targets of Ccr4-Not involved in controlling transcription elongation. The long term goal of this project is to understand how elongation factors assist RNAPII in transcribing genes and maintaining the integrity of the genome of eukaryotic cells.
Numerous human diseases and syndromes are caused by disturbances in gene expression, and an important component in this process is the ability of cells to complete the production of mRNAs from the genome. The goal of the work described in this proposal is to understand how protein complexes called elongation factors allow for the efficient production of functional mRNAs. The complexes that are the focus of this proposal have been implicated in DNA damage resistance, cell proliferation and cardiovascular development; thus, the work described here is directly relevant to human health.
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