Structure/Function of Transcription Complex Regulation

转录复合物调控的结构/功能

• 批准号: 7988507
• 负责人: Robert Landick
• 金额: $ 9.76万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988507
• 关键词: Active Sites; Affect; Bacteria; Bacterial Proteins; Biochemical Genetics; Biochemistry; Biological Models; Biophysics; Catalysis; Cities; Complex; DNA; DNA-Directed RNA Polymerase; Development; Dissection; Escherichia coli; Eukaryota; Event; Face; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Grant; Growth; HIV; HIV-1; Human; Human Resources; Instruction; Kinetics; Lateral; Location; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Modeling; Movement; Names; New York; Nucleotides; Principal Investigator; Printing; Proteins; RNA; RNA Polymerase II; Regulation; Research Personnel; Research Project Grants; Resistance; Role; SHPS-1 protein; Signal Transduction; Signaling Protein; Site; Structure; Surgical Flaps; Testing; Transcript; Transcription Elongation; Transcriptional Regulation; Universities; Virus; Wisconsin; Work; farmer; genetic regulatory protein; premature; programs; response

The long-range goal of this project is to define the interactionsin the transcriptioncomplex that regulate pausing and termination by RNA polymerase. Nascent RNA hairpins are important regulatory signals in bacteria, where pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect expression of genes in mammalian cells and viruses, notably genes involved in the development of cancer and in growth of the AIDS virus, HIV-1. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcriptioncomplex to make it resistant to pausing and termination. Although significant progress has been made in understandingpausing, termination, and the regulatory proteins that control these events, two alternativemodels remain possible. In one view, called the allosteric model, pause signals, termination signals, and regulatory proteins primarily affect the ¿conformation of RNA polymerase. In the other, these signals and proteins primarily affect translocation of a relatively rigid RNA polymerase on the RNA and DNA chains (the rigid-body model). Pausing and termination by E. coli RNA polymerase and their regulation by the NusA, NusG, and RfaH proteins, and pausing by human RNA polymerase II have been developed as model systems. A combination of biochemical, genetic, and biophysical approaches will be used to distinguish the allosteric and rigid-body models of transcriptional regulation, and to characterize the mechanisms of pausing, termination, and regulatory proteins that control them. Specific aims will be to (i) characterize interactions of RNA polymerase's flap-tip helix with RNA, NusA, and a70, and test how these interactions affect catalysis in the active site; (ii) determine the location of the RNA 3' end in paused and nonpaused transcription elongation complexes; (Hi)determine the kinetic mechanisms of elongation, pausing, and termination; (iv) map interactions between RNA polymerase and pause and terminator hairpins; and (v) determine the sites at which RfaH and NusG interact with RNA polymerase and the mechanisms by which they regulate transcript elongation. E si Ib(S) (organization, city, state) University of Wisconsin, Madison, WI Rockefeller University, New York, NY Brandeis University, Waltham, MA Stanford University, Stanford, CA St. Louis University, St. Louis, MO KEY PERSONNEL. See instructions on Page 11. Name Robert Landick Irina Artsimovitch Innokenti Toulokhonov Murali Palangat Kati Geszvain Christina Farmer Rachel Mooney Seth Darst JeffGelles Steve Block Tomasz Heyduk Use continuation pages as needed to provide the required information in the format shown below. Organization Role on Project University of Wisconsin-Madison Principal Investigator University of Wisconsin-Madison Postdoctoral Researcher University of Wisconsin-Madison Postdoctoral Researcher University of Wisconsin-Madison Postdoctoral Researcher University of Wisconsin-Madison Graduate Assistant University of Wisconsin-Madison Graduate Assistant University of Wisconsin-Madison Graduate Assistant Rockefeller University Collaborator Brandeis University Collaborator Stanford University Collaborator St. Louis University Collaborator PHS 398 (Rev. 4/98) Page 2 BB CC Principal Invesjjaator/Program Director (Last, first, middle): Roherf T .andirlr Type the name of the principal investigator/prograi^Hbctor at the top of each printed page and each con^Btion page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,

这个项目的长期目标是确定转录复合体中的相互作用， 通过RNA聚合酶暂停和终止。新生RNA发夹是重要的调控信号， 细菌，其中暂停和终止是遗传调节机制的主要组成部分。暂停 和过早终止也会影响哺乳动物细胞和病毒中的基因表达， 参与癌症的发展和艾滋病病毒HIV-1的生长。在细菌和 在真核生物中，专门的调节蛋白修饰转录复合体，使其抵抗暂停， 和终止。虽然在理解暂停、终止和 控制这些事件的调节蛋白，两个替代模型仍然是可能的。一种观点认为， 称为变构模型，暂停信号，终止信号和调节蛋白主要影响 RNA聚合酶的构象。在另一方面，这些信号和蛋白质主要影响细胞的易位。 RNA和DNA链上相对刚性的RNA聚合酶（刚体模型）。暂停和 终止E。大肠杆菌RNA聚合酶及其受NusA、NusG和RfaH蛋白的调节，以及 通过人RNA聚合酶II的暂停已经被开发为模型系统。 生物化学、遗传学和生物物理学方法的组合将用于区分 转录调控的变构和刚体模型，并表征 暂停、终止和控制它们的调节蛋白。具体目标是：（一） RNA聚合酶的瓣尖螺旋与RNA，NusA和a70的相互作用，并测试这些相互作用 影响活性位点的催化作用;（ii）确定RNA 3'端在暂停和非暂停中的位置 转录延伸复合物;（iii）确定延伸、暂停和转录的动力学机制。 终止;（iv）绘制RNA聚合酶与暂停和终止子发夹之间相互作用;和（v） 确定RfaH和NusG与RNA聚合酶相互作用的位点以及 它们调节转录延长。 E si Ib（S）（组织、城市、州） 威斯康星州，麦迪逊，威斯康星州 洛克菲勒大学，纽约州纽约 Brandeis University，Waltham，MA 斯坦福大学，加利福尼亚州，斯坦福大学 圣路易斯大学，密苏里州圣路易斯 关键人员。参见第11页的说明。 名称 罗伯特·兰迪克 伊琳娜·阿尔齐莫维奇 因诺肯季·图洛霍诺夫 穆拉利·帕兰加特 卡蒂·格兹万 克里斯蒂娜·法默 雷切尔·穆尼 塞斯·达斯特 杰夫·盖尔斯 史蒂夫·布洛克 托马斯·海杜克 根据需要使用续页，以下列格式提供所需信息。 组织在项目中的角色 威斯康星大学麦迪逊分校首席研究员 威斯康星大学麦迪逊分校博士后研究员 威斯康星大学麦迪逊分校博士后研究员 威斯康星大学麦迪逊分校博士后研究员 威斯康星大学麦迪逊分校研究生助理 威斯康星大学麦迪逊分校研究生助理 威斯康星大学麦迪逊分校研究生助理 洛克菲勒大学合作者 布兰迪斯大学合作者 斯坦福大学合作者 圣路易斯大学合作者 PHS 398（Rev. 4/98）第2 BB页 CC首席研究员/项目总监（最后，第一，中间）：Roherf T .andirlr 在各打印页和各附页的顶部键入主要研究者/研究者姓名。(For型号规格，见 第6页的说明）。 研究资助 目录 页码 首页1 产品描述：