CONTROL OF RIBOSOMAL GENE TRANSCRIPTION

这个项目继续我们长期以来的调查， 真核生物核糖体RNA合成调控的分子基础 细胞 我们最近改用酵母作为实验生物 拟议的研究分为四大类： 1)我们将使用各种遗传学方法继续 基因鉴定和部分蛋白质的cDNA克隆 RNA利用的基础转录起始机制 聚合酶I（Poll）。这些遗传工具包括一种新的殖民地颜色 我们已经开发了一种检测方法，其中酵母菌菌落从暗红色变为 淡粉色，因为Poll转录被激活。 2)Pol I转录活性与细胞的整体生长密切相关， 细胞的速率。我们将结合基因和生化技术 方法来识别和研究信号通路，导致从 细胞的外部连接到Poll机器。 3)我们之前已经从青蛙中克隆了Poll转录因子， 非洲爪蟾，称为xUBF。我们将继续研究这是如何 蛋白质结合到DNA，核小体，以及它如何作为一个 转录因子与巴里斯托达德博士合作，我们将 使xUBF的衍生物结晶并确定其三维结构 由X射线晶体学构造。基因和生化方面 方法将用于鉴定xUBF的推定酵母同系物。 4)生物化学破碎酵母全细胞的持续努力 提取将继续进行。一些转录因子可以被鉴定 并通过这条路线进行克隆。此外，一个良好的分馏系统， 对于详细分析分子机制至关重要， 调节Poll转录。 核糖体RNA占典型细胞中总RNA的一半以上， 重要的是要了解这种主要细胞的合成 组件是规范的。不过，目前尚不清楚这是否 这些知识将立即具有临床实用性。

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.