Human RNAPII Structure/Function in Pausing & Elongation

描述(申请人提供)：这个项目的长期目标是定义转录复合体中的相互作用，允许人类RNA聚合酶II调节暂停和终止。暂停和终止是专门的调节蛋白的靶标，它修改转录复合体，允许合成完整的mRNA链。这些调节蛋白改变RNA聚合酶II活性的机制尚不清楚。为了提供一个概念框架，在其中可以定义这些机制，我们正在阐明来自HIV-1早期转录区域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/功能研究需要获得具有特定改变的氨基酸序列的人RNA聚合酶II酶的能力。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质作用的机制。我们现在已经建立了一种方法，可以用来生产重组突变的人RNA聚合酶II，并且手头有一种突变酶，这种酶在伸长方面存在缺陷。这一突变酶和其他提出的突变酶不仅有助于研究停顿的基本机制，还有助于研究调节停顿的蛋白质因素之间的相互作用。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)剖析负延长因子的作用机制；以及(Iii)使用标记的RNA聚合酶II对人类RNA聚合酶II的转录延长进行单分子分析。众所周知，人类RNA聚合酶II的非调控转录延长是导致艾滋病和某些癌症等致命人类疾病的主要因素。然而，关于RNA聚合酶II在此类疾病中的伸长特性如何改变，人们知之甚少。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.