TERMINATION OF TRANSCIRPTION BY RNA POLYMERASE I

我们将继续探索转录所涉及的机制 酿酒酵母中核糖核酸聚合酶L的终止作用。 目前，我们知道高效的最小DNA序列 在体外终止，我们知道一种名为Reblp的蛋白质与此结合 序列，并作为终结者蛋白，我们有证据表明 RNA3‘处理活动与终止有关。我们已经提议 一种结合了所有这些元素的终止模式，我们 将以尽可能多的方式测试这一模型。 为了将来的研究，我们已经开发了一种体内颜色分析方法，其中 当POLI终止处于活动状态时，酵母菌落从白色变为红色。 使用这种分析方法，我们将更严格地定义DNA序列和 在活细胞中终止所需的蛋白质。这将涉及到 对参与终止的蛋白质进行基因筛查以及 进一步定义已知蛋白质的区域(如Poli本身和 Reblp)，这对于终止是必不可少的。 与轮询相关的RNA3‘端处理活动类似于 作用，但在物理上不同于PolII伸长系数Sii (TFIIS)。我们将纯化POLI相关加工因子，克隆其 基因，并确定其在终止过程中的作用(如果有)。 投票终止模型的一个基本部分是这样一个命题 与其DNA位点结合的Reblp充当一种暂停信号，该信号专用于 投票。为了确定这是如何发生的，我们打算确定晶体 与ITS共结晶的Reblp DNA结合域的结构 同源DNA站点。 使用差动终止和/或块伸长作为对照 许多人类癌基因以及艾滋病毒-1的机制。目前 轮询终止可能是最容易理解和最容易获得的 真核RNA聚合酶的终止系统。我们相信 在这个模型系统中获得的见解可能有助于理解 也可以被其他真核RNA聚合酶终止。

We will continue to explore the mechanisms involved in transcription termination by RNA polymerase l in the yeast, Saccharomyces cerevisiae. At present we know the minimal DNA sequences needed for efficient termination in vitro, we know that a protein, Reblp, binds to this sequence and acts as a terminator protein, and we have evidence that an RNA 3' processing activity associates with termination. We have proposed a model for termination that incorporates all of these elements and we will test this model in as many ways as possible. For future studies we have developed an in vivo color assay in which yeast colonies change from white to red when polI termination is active. Using this assay we will more rigorously define both DNA sequences and proteins required for termination in the living cell. This will involve doing genetic screens for proteins involved in termination as well as further defining the regions of known proteins (such as polI itself and Reblp) which are essential for termination. The RNA 3' end processing activity associated with poll is similar in action but physically distinct from the polII elongation factor, SII (TFIIS). We will purify the polI associated processing factor, clone its gene, and determine its role, if any, in the termination process. An essential part of the poll termination model is the proposition that Reblp bound to its DNA site acts as a pausing signal that is specific for poll. To determine how this occurs we intend to determine the crystal structure of the Reblp DNA binding domain co-crystallized with its cognate DNA site. Differential termination and/or blocks to elongation are used as control mechanisms for numerous human oncogenes as well as for HIV-1. At present poll termination is probably the best understood and most accessible termination system for eukaryotic RNA polymerases. We believe that insights gained in this model system will likely aid in understanding termination by other eukaryotic RNA polymerases as well.