This project continues our long standing investigation into the molecular basis for regulating ribosomal RNA synthesis in eukaryotic cells. We have recently switched to yeast as our experimental organism and the proposed studies fall under four broad headings: 1) We will use a variety of genetic approaches to continue identification of genes and cloning of cDNAs for proteins that are part of the basal transcription initiation machinery utilized by RNA polymerase l (Poll). These genetic tools include a novel colony color assay we have developed in which yeast colonies change from dark red to light pink as Poll transcription is activated. 2) Pol I transcription activity is closely tied to the overall growth rate of the cell. We will use a combination of genetic and biochemical approaches to identify and study the signalling pathway which leads from the exterior of the cell to the Pol l machinery. 3) We have previously cloned a Poll transcription factor from the frog, Xenopus laevis, called xUBF. We will continue our studies of how this protein binds to DNA, to nucleosomes, and how it functions as a transcription factor. In collaboration with Dr. Barry Stoddard we will crystallize derivatives of xUBF and determine its 3-dimensional structure by X-ray crystallography. Both genetic and biochemical approaches will be used to identify the putative yeast homolog of xUBF. 4) Ongoing efforts to biochemically fractionate a yeast whole cell extract will be continued. Some transcription factors may be identified and cloned by this route. In addition, a well fractionated system is essential for detailed analysis of the molecular mechanisms that regulate Poll transcription. Ribosomal RNA comprises over half of the total RNA in a typical cell and it seems important to understand how synthesis of such a major cellular component is regulated. However, at present it is not clear whether this knowledge will have immediate clinical utility.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Cytology Study Section (CTY)
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Fred Hutchinson Cancer Research Center
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Aprikian, P; Moorefield, B; Reeder, R H (2001) New model for the yeast RNA polymerase I transcription cycle. Mol Cell Biol 21:4847-55
Aprikian, P; Moorefield, B; Reeder, R H (2000) TATA binding protein can stimulate core-directed transcription by yeast RNA polymerase I. Mol Cell Biol 20:5269-75
Moorefield, B; Greene, E A; Reeder, R H (2000) RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human. Proc Natl Acad Sci U S A 97:4724-9
Lin, C W; Moorefield, B; Payne, J et al. (1996) A novel 66-kilodalton protein complexes with Rrn6, Rrn7, and TATA-binding protein to promote polymerase I transcription initiation in Saccharomyces cerevisiae. Mol Cell Biol 16:6436-43
Schultz, M C; Choe, S Y; Reeder, R H (1993) In vitro definition of the yeast RNA polymerase I enhancer. Mol Cell Biol 13:2644-54
Palmer, T D; Miller, A D; Reeder, R H et al. (1993) Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter. Nucleic Acids Res 21:3451-7
Schultz, M C; Brill, S J; Ju, Q et al. (1992) Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation. Genes Dev 6:1332-41
Schultz, M C; Choe, S Y; Reeder, R H (1991) Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc Natl Acad Sci U S A 88:1004-8
McStay, B; Hu, C H; Pikaard, C S et al. (1991) xUBF and Rib 1 are both required for formation of a stable polymerase I promoter complex in X. laevis. EMBO J 10:2297-303
McStay, B; Frazier, M W; Reeder, R H (1991) xUBF contains a novel dimerization domain essential for RNA polymerase I transcription. Genes Dev 5:1957-68

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