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.
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