TRANSCRIPTION OF RAT RIBOSOMAL DNA

了解编码的基因的转录机制 如果我们要理解正常的和非正常的 异常生长过程，例如伤口愈合和肿瘤形成。核糖体 生物发生，因此核糖体RNA基因的表达，是 与细胞生长速度相协调，并对各种 信号，取决于所研究的细胞类型。的长期目标是 我们的研究是为了确定核糖体基因(S) 转录是受调控的。脊椎动物核糖体RNA的启动子 基因(RDNA)具有相似的功能元件，尽管它们的核苷酸 序列通常是完全不同的。此外，既有保守的， 和同源但特定物种的反式作用因子，以及 利用这些同源因子的物种特定模式。这个 哺乳动物rDNA启动子由两个广泛定义的元件组成：核心 启动子元件(CPE)和上游启动子元件(UPE)。CPE是 对于体外转录来说是必要的和充分的，对于 体内转录。然而，UPE是转录所必需的 体内，以及在体外高效转录，以及 形成稳定的预引发复合体。至少有两个变装演员 RNA准确高效转录所需的因子 聚合酶I、SL-L和UBF。转写需要SL-L，而UBF 激活转录。我们已经纯化了大鼠SL-L，所需的因子 进行物种特异性转录，并确定它与 CPE和UPE。我们已经纯化并克隆了大鼠的UBF。纯化的大鼠 UBF，由两个多肽组成，足迹在-120到-50之间 大鼠rDNA启动子，在被定义为UPE的结构域内。然而，对于 UBF对体外转录的影响，SL-L在UPE中的结合部位 必须完好无损。此外，我们还发现，这两种多肽存在 UBF制剂中可能是两个独立的mRNAs的产物，并且 它们是密切相关的蛋白质，可能具有重要意义 DNA结合或功能上的差异。这一假设将继续下去。 大鼠SL-L的纯化和鉴定，以及UBF的具体作用 并对转录中的SL-L进行了界定。此外，我们的结果表明， 至少还需要一个其他因素才能形成稳定的 预引发复合体，我们将提纯该因子。通过合并在 高纯度体外转录和DNA结合分析 转录因子与体内启动子的各种突变体 表达分析，我们将确定转录的机制 已经完成了。拟议中的实验将增加我们对 这个多基因家族的转录机制，以及 调控其转录的机制。

Understanding the mechanisms of transcription of the genes that code for 45S pre-ribosomal RNA is essential if we are to understand both normal and abnormal growth processes, e.g. wound healing and neoplasia. Ribosome biogenesis, and therefore the expression of the ribosomal RNA genes, is coordinated with the rate of cell growth, and responds to a variety of signals, depending upon the cell type studied. The long-term objective of our research is to determine the mechanism(s) by which ribosomal gene transcription is regulated. The promoters of vertebrate ribosomal RNA genes (rDNA) have analogous functional elements, although their nucleotide sequences are often quite different. Further, there are both conserved, and homologous but species-specific trans-acting factors, as well as species-specific patterns of utilization of these homologous factors. The mammalian RDNA promoters consist of two broadly defined elements: a core promoter element (CPE) and an upstream promoter element (UPE). The CPE is necessary and sufficient for transcription in vitro, and necessary for transcription in vivo. However, the UPE is required for transcription in vivo, and for efficient transcription in vitro, as well as for the formation of a stable preinitiation complex. At least two trans-acting factors are required for accurate and efficient transcription by RNA polymerase I, SL-l and UBF. SL-l is required for transcription, while UBF activates transcription. We have purified rat SL-l, the factor required for species-specific transcription, and determined that it interacts both the CPE and the UPE. We have purified and cloned rat UBF. Purified rat UBF, which consists of two polypeptides, footprints between -120 and -50 of the rat rDNA promoter, within the domain defined as the UPE. However, for UBF to affect transcription in vitro, the binding site for SL-l in the UPE must be intact. Moreover, we have found that the two polypeptides present in preparations of UBF are probably the products of two separate mRNAs, and that they are closely related proteins with what may be significant differences in DNA-binding or function. This hypothesis will be pursued. Rat SL-l will be purified and characterized, and the specific roles of UBF and SL-l in transcription will be defined. Further, our results indicate that at least one other factor is required for the formation of a stable preinitiation complex, and we will purify that factor. By combining in vitro transcription and DNA-binding assays using highly purified transcription factors and various mutants of the promoter with in vivo expression assays, we will determine the mechanism by which transcription is accomplished. The proposed experiments will increase our understanding of the mechanism of transcription of this multi-gene family, and the mechanism by which its transcription is regulated.