Human RNAPII Structure/Function in Pausing & Elongation

暂停中的人类 RNAPII 结构/功能

• 批准号: 7211464
• 负责人: Robert Landick
• 金额: $ 19.23万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7211464
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Affect; Amanitins; Amino Acid Sequence; Binding; Binding Sites; Biochemistry; Biological Assay; Complex; DNA; DNA Sequence; DNA Sequence Rearrangement; Disease; Dissection; Elongation Factor; Enzymes; Event; Genetic Transcription; Goals; HIV-1; Hand; Helix (Snails); Human; Hybrids; Knowledge; Life; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Methods; Modeling; Molecular Biology; Movement; Nucleic Acids; POLR2A gene; Personal Satisfaction; Property; Proteins; RNA; RNA Polymerase II; Range; Recombinants; Regulation; Research; Role; Signal Transduction; Site; Structure; Study models; Surgical Flaps; System; Testing; Transcript; Transcription Elongation; Work; Yeasts; crosslink; ear helix; elongin; genetic regulatory protein; human POLR2A protein; human disease; inhibitor/antagonist; mutant; negative elongation factor; research study; response; single molecule; transcription factor S-II; transcription factor TFIIF

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.

描述（由申请人提供）：该项目的远程目标是定义转录复合物中的相互作用，以调节人RNA聚合酶II的暂停和终止。暂停和终止是专门的调节蛋白的靶标，可修饰转录复合物以合成完整的mRNA链。这些调节蛋白改变RNA聚合酶II活性的机制尚不清楚。为了提供一个可以定义这些机制的概念框架，我们正在阐明来自HIV-1早期转录区域的模型暂停信号的基本机制。在结构细节中定义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对暂停信号的响应。但是，暂停机制的结构/功能研究需要具有特异性改变氨基酸序列的人RNA聚合酶II酶的能力。该项目的总体目标是制造几种人RNA聚合酶II突变体，并使用它们来测试转录暂停模型和调节蛋白作用的机制。现在，我们已经建立了一种方法，可以通过这种方法产生重组突变的人RNA聚合酶II，并在手头上具有伸长率有缺陷的突变酶。这种和其他提出的突变酶不仅可以研究暂停的基本机制，而且还可用于调节暂停的蛋白质因子的相互作用。该项目的具体目的是（i）使用改变的人RNA聚合酶II酶来定义暂停和调节蛋白质作用的机制； （ii）剖析负伸长因子的作用机理； （iii）使用标记的RNA聚合酶II进行人RNA聚合酶II的转录伸长分子分析。众所周知，人RNA聚合酶II不受管制的转录伸长是艾滋病和某些癌症等致命人类疾病的主要因素。然而，对于这种疾病中RNA聚合酶II的伸长特性如何改变了，知之甚少。我们的研究旨在理解这些转录伸长的基本机制。获得的知识将增加对人类疾病基本分子生物学的理解。