Grant: $82,339 - National Institutes of Health - Sep. 9, 2009
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Award Description: The elongation of transcripts by RNA polymerase II is a complex process dependent upon nucleotide substrate levels, elongation factors, and co-transcriptional events such as splicing. Mutations in genes encoding elongation factor SI I (TFIIS), other elongation factors, RNA polymerase subunits, and chromatin related proteins, render S. cerevisiae sensitive to the drugs 6-azauracil and mycophenolic acid. These drugs are inhibitors of IMP dehydrogenase (IMPDH) which is a critical enzyme in the de novo pathway of guanine nucleotide synthesis. Its abundance is positively correlated with cell growth in eukaryotes from yeast to humans. Drug treated yeast respond by transcriptional induction of one of their four IMPDH genes. The response is reliant upon an optimally functioning elongation machinery. In humans, T lymphocyte proliferation during an immune response is highly dependent upon IMPDH synthesis. Mycophenolic acid inhibits T cell proliferation and is an important clinical immunosuppressant that inhibits graft rejection in kidney transplant (and other) patients. Mutations in IMPDH have also been implicated in the etiology of retinitis pigmentosa. Regulation of the essential yeast IMPDH gene family is mediated through a cellular mechanism that senses nucleotide pools. This project will use biochemistry and genetics to understand this regulation of IMPDH transcription through novel DNA elements. One sequence is a potent repressive element which encompasses the transcription start site and may regulate elongation. Yeast contain a complex family of IMPDH isoforms with varying sensitivity to mycophenolic acid inhibition. The potential of IMPDH to form heteromeric complexes between drug resistant and sensitive subunits will be addressed. The drug sensitive phenotype of yeast lacking their IMPDH genes will be useful in studying resistant and sensitive forms in humans. Resistance to IMPDH-directed drugs is an important clinical issue given the wide spread and lifelong treatment of transplant patients with mycophenolate. The ability of normal T cells, and T cells from patients receiving therapy, to respond to the drug by altering IMPDH expression and activity, will be examined. Differences between the responses of different individuals will be quantified. This will be important for the long term goal of understanding potential genetic differences in the efficacy of therapy and responsiveness of patients to immunosuppressants that target IMPDH.
Project Description: This supplement requests the support of laboratory staff to maintain personnel and momentum of a project that focuses on transcriptional regulation in S. cerevisiae. The IMD2 gene in this yeast responds to the level of intracellular guanine nucleotides to govern the production of its gene product, which in turn is the rate limiting enzyme for guanine nucleotide synthesis. Recently, a novel regulatory mechanism for this gene has been uncovered that involves alternative start site selection, transcription of non-coding labile RNAs, and premature termination. The control strategy for IMD2 is the prototype of what has become a growing number of genes governed by decisions regarding transcription start site selection and non-coding RNAs. The regulated termination event employs RNA binding proteins, RNA polymerase II-binding proteins, and the essential protein Sen1. This project will examine the biochemistry of Sen1, a protein which is a putative helicase and for which there is a human disease-related ortholog. We will directly test recombinant Sen1 for RNA binding, nucleic acid-dependent ATPase, and helicase activity to discern which of these activities is essential in living cells. The human version of the protein, senataxin, willl also be examined for helicase activity. Altered versions of the protein will be generated that correspond to yeast mutants known to compromise growth and human mutants that are causally implicated in disease etiology to learn if these problems are related to Sen1's helicase activity.
Infrastructure Description: N/A
Jobs Summary: N/A (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on Sep. 9, 2009. Help expand these official descriptions using the wiki below.