Human RNAPII Structure/Function in Pausing & Elongation

描述（由申请人提供）：该项目的长期目标是确定转录复合体中允许人类RNA聚合酶II暂停和终止调控的相互作用。暂停和终止是专门调节蛋白的目标，这些蛋白修饰转录复合体以合成完整的mRNA链。这些调节蛋白改变RNA聚合酶II活性的机制尚不清楚。为了提供一个定义这些机制的概念框架，我们正在阐明来自HIV-1早期转录区的模型暂停信号的基本机制。在结构细节上定义HIV-1暂停机制将使我们能够确定调节蛋白如何影响RNA聚合酶II对暂停信号的反应。然而，暂停机制的结构/功能研究需要能够获得具有特异性改变的氨基酸序列的人类RNA聚合酶II酶。该项目的总体目标是制造几个人类RNA聚合酶II突变体，并用它们来测试转录暂停模型和调节蛋白作用的机制。我们现在已经建立了一种方法，通过这种方法我们可以生产重组突变的人类RNA聚合酶II，并且手头有一个突变的酶是有缺陷的延伸。这种和其他提出的突变酶不仅有助于研究暂停的基本机制，而且有助于研究调节暂停的蛋白质因子的相互作用。该项目的具体目标是：(i)使用改变的人类RNA聚合酶II酶来确定暂停和调节蛋白质作用的机制；（ii）剖析负延伸因子的作用机制；（iii）使用标记RNA聚合酶II进行人RNA聚合酶II转录延伸的单分子分析。众所周知，人类RNA聚合酶II不受调节的转录延伸是艾滋病和某些癌症等致命人类疾病的主要因素。然而，对于RNA聚合酶II的延伸特性在这些疾病中是如何改变的，我们知之甚少。我们的研究旨在了解这些转录延伸的基本机制。所获得的知识将增加对人类疾病的基本分子生物学的理解。

DESCRIPTION (provided by applicant): The long-range goal of this project is to define the interactions in the transcription complex that allow regulation of pausing and termination by human RNA polymerase II. Pausing and termination are the targets of specialized regulatory proteins that modify the transcription complex to allow synthesis of complete mRNA chains. The mechanisms by which these regulatory proteins alter the activity of RNA polymerase II are unknown. To provide a conceptual framework within which these mechanisms can be defined, we are elucidating the fundamental mechanism of a model pause signal from the early transcribed region of HIV-1. Defining the HIV-1 pause mechanism in structural detail will then allow us to determine how regulatory proteins affect the response of RNA polymerase II to the pause signal. However, a structure/function study of pause mechanisms requires the ability to obtain human RNA polymerase II enzymes with specifically altered amino-acids sequences. The overall goal of the project is to make several human RNA polymerase II mutants and use them to test models of transcriptional pausing and the mechanisms of regulatory protein action. We now have established a method by which we can produce recombinant mutant human RNA polymerase II, and have on hand a mutant enzyme that is defective for elongation. This and other proposed mutant enzymes will be useful to study not only the basic mechanism of pausing, but also the interactions of protein factors that regulate pausing. The specific aims of the project are (i) use altered human RNA polymerase II enzymes to define mechanism of pausing and regulatory protein action; (ii) dissect the mechanism of action of a negative elongation factor; and (iii) use tagged RNA polymerase II for single molecule analysis of transcription elongation by human RNA polymerase II. It is well established that unregulated transcription elongation by human RNA polymerase II is a major factor in deadly human diseases like AIDS and certain cancers. However, little is known about how the elongation properties of RNA polymerase II are altered in such diseases. Our research is aimed at understanding these fundamental mechanisms of transcription elongation. The knowledge gained will increase understanding of the basic molecular biology of human diseases.