TERMINATION OF TRANSCIRPTION BY RNA POLYMERASE I

RNA 聚合酶 I 终止转录

基本信息

批准号：
2608893
负责人：
RONALD H REEDER
金额：
$ 31.85万
依托单位：
FRED HUTCHINSON CANCER RESEARCH CENTER
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-12-01 至 1999-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2608893
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

项目摘要

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.

我们将继续探索涉及转录的机制 RNA聚合酶L在酵母，酿酒酵母中终止。目前，我们知道有效的最小DNA序列在体外终止，我们知道蛋白质REBLP与此结合序列并充当终结蛋白，我们有证据表明 RNA 3'处理活性与终止相关。我们提出了结合所有这些要素的终止模型，我们将以尽可能多的方式测试此模型。对于未来的研究，我们开发了一种体内颜色测定法当poli终止活性时，酵母菌菌落从白色变为红色。使用此测定法，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。这将涉及为参与终止的蛋白质进行遗传筛选以及进一步定义已知蛋白质的区域（例如poli本身和 REBLP），这对于终止至关重要。与民意调查相关的RNA 3'末端处理活动在作用，但实际上与polii伸长因子Sii不同（tfiis）。我们将净化poli相关的处理因子，克隆其基因，并确定其在终止过程中的作用（如果有）。民意调查终止模型的重要组成部分是一个主张与其DNA位点结合的REBLP充当特定的暂停信号轮询。为了确定这种情况的发生方式，我们打算确定晶体 REBLP DNA结合结构域的结构与其共结晶及其结构同源DNA位点。差终止和/或伸长块用作控制许多人类癌基因以及HIV-1的机制。现在民意测验终止可能是最能理解的，最容易获得的真核RNA聚合酶的终止系统。我们相信在此模型系统中获得的见解可能有助于理解其他真核RNA聚合酶也终止。