TRANSCRIPTION OF RAT RIBOSOMAL DNA

了解基因的转录机制， 45 S前核糖体RNA是必不可少的，如果我们要了解正常和 异常生长过程，例如伤口愈合和瘤形成。 核糖体 生物发生，因此核糖体RNA基因的表达， 与细胞生长的速度相协调，并对各种 信号，这取决于所研究的细胞类型。 的长期目标 我们的研究是确定核糖体基因 转录是受调控的。 脊椎动物核糖体RNA启动子 基因（rDNA）具有类似的功能元件，尽管它们的核苷酸 序列通常是完全不同的。 此外，两者都是保守的， 和同源但物种特异性的反式作用因子，以及 这些同源因子的物种特异性利用模式。 的 哺乳动物rDNA启动子由两个广泛定义的元件组成： 启动子元件（CPE）和上游启动子元件（UPE）。 所述用户驻地设备 必要和充分的体外转录，必要的 体内转录。 但是，在以下情况下需要UPE进行转录： 体内，并在体外有效转录，以及为 形成稳定的起始前复合物。 至少两个反式作用 这些因子是RNA准确有效转录所必需的 聚合酶I、SL-1和UBF。 SL-1是转录所必需的，而UBF 激活转录。 我们已经纯化了大鼠SL-1， 物种特异性转录，并确定它相互作用， CPE和UPE。 我们已经纯化并克隆了大鼠UBF。 纯化的大鼠 UBF由两种多肽组成，足迹在-120和-50之间， 大鼠rDNA启动子，在定义为UPE的结构域内。 但对于 UBF影响体外转录，UPE中SL-1的结合位点 必须完好无损 此外，我们发现，存在的两种多肽 在UBF的制备中，可能是两种单独的mRNA的产物， 它们是密切相关的蛋白质， DNA结合或功能的差异。 我们将继续探讨这一假设。 本实验将对大鼠SL-1进行纯化和鉴定，并对UBF的特异性作用进行初步研究。 和转录中的SL-1将被定义。 此外，我们的研究结果表明， 至少需要一个其他因素来形成稳定的 启动前复合体，我们将纯化该因子。 通过结合 体外转录和DNA结合试验，使用高度纯化的 转录因子和启动子的各种突变体， 表达测定，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 它的转录调控机制。

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.