TERMINATION OF TRANSCIRPTION BY RNA POLYMERASE I

RNA 聚合酶 I 终止转录

• 批准号: 2022295
• 负责人: RONALD H REEDER
• 金额: $ 30.84万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022295
• 关键词: DNA binding protein; DNA directed RNA polymerase; Saccharomyces cerevisiae; crystallization; genetic promoter element; laboratory rabbit; molecular cloning; molecular genetics; nucleic acid sequence; polymerase chain reaction; posttranscriptional RNA processing; protein structure; transcription factor; transcription termination

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.

我们将继续探索转录所涉及的机制 在酵母，酿酒酵母中通过RNA聚合酶1终止。 目前，我们知道高效的DNA序列需要的最小的DNA序列， 在体外终止，我们知道一种蛋白质，Reblp， 序列，并作为终止蛋白，我们有证据表明， RNA 3'加工活性与终止相关。我们提出 一个包含所有这些元素的终止模式， 将尽可能多地测试这个模型 对于未来的研究，我们已经开发了一种体内颜色测定法，其中 当polI终止激活时，酵母菌落从白色变为红色。 使用该测定，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 对参与终止的蛋白质进行基因筛选， 进一步定义已知蛋白质的区域（如polI本身， Reblp），这对于终止至关重要。 与poll相关的RNA 3'末端加工活性与 作用，但在物理上不同于polII延长因子，SII （TFIIS）。我们将纯化polI相关的加工因子，克隆其 基因，并确定其作用，如果有的话，在终止过程中。 轮询终止模型的一个基本部分是这样一个命题， Reblp结合到其DNA位点上，作为一种暂停信号， 民调 为了确定这是如何发生的，我们打算确定晶体 Reblp DNA结合结构域的结构与其共结晶 同源DNA位点。 差异终止和/或伸长阻断用作对照 许多人类致癌基因以及HIV-1的机制。目前 轮询终止可能是最好理解和最容易理解的 真核RNA聚合酶终止系统。 我们认为 在这个模型系统中获得的见解可能有助于理解 其他真核生物RNA聚合酶的终止。