RNA BIOSYNTHESIS IN ESCHERICHIA COLI

大肠杆菌中的 RNA 生物合成

• 批准号: 2838605
• 负责人: Alexander Goldfarb
• 金额: $ 29.44万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838605
• 关键词: DNA directed RNA polymerase; Escherichia coli; RNA; active sites; affinity labeling; autoradiography; bacterial genetics; chromosome translocation; crosslink; enzyme activity; gene expression; genetic mapping; genetic promoter element; genetic transcription; high performance liquid chromatography; immobilized enzymes; nucleic acid biosynthesis; nucleic acid hybridization; nucleic acid probes; protein structure function; radiotracer; rifamycins; transcription factor

DESCRIPTION: The primary focus of the research will be on three very important features of prokaryotic transcription elongation: (i) processivity; (ii) the relationship between RNAP movement and nucleotide addition; (iii) fidelity and error correction. The work will be based on a new model for elongation recently proposed by Dr. Goldfarb, which constitutes a revision to the previously held "inchworm" model. The main idea will be to use chemical crosslinking and protein chemistry to define the contacts made by different functional components or RNAP, including the front-end DNA binding site (DBS), the catalytic active center (AC), the RNA-DNA hybrid binding site (HBS), and the upstream RNA product binding site (RBS). In addition, the investigator proposes to dissect the basic nucleotide addition reaction into pre- and post-translocation states. During the past five years, a number of innovative techniques have been developed, primarily in the Goldfarb laboratory, which provide sophisticated tools for the investigation of structure-function relationships in elongation: (i) RNAP walking experiments are be done with His-tagged enzyme chelated to Ni-agarose beads, thus permitting the isolation of defined complexes at each step. (ii) Crosslinking probes can be incorporated into RNA at the 5' and 3' ends or within the body of the transcript, as well as in any position on the DNA template. These can then be used to demonstrate which parts of RNAP are contacted by the tagged nucleotide, and have also been used to demonstrate the formation and extent of the nascent RNA-DNA hybrid. (iii) Derivatized crosslinkable rifampicin compounds have been used to identify the relationship between the rif binding site and the transcription startsite. (iv) Fe++ -induced cleavage has helped identify sites in the enzyme that make up the active center (AC). A number of these methods have been used to characterize the active center, which appears to be made up of modules coming from distinct regions of both beta and beta'. The basic design of the proposed experiments will be to use specific radioactive crosslinking agents substituted at particular positions in the nascent RNA or in the DNA template, followed by cleavage of the crosslinked protein subunit with any of several different amino acid-specific cleavage reagents. Various crosslinkers with different crosslinking radii, or with the ability to be placed at different positions in nascent RNA, will be employed Many of these have been developed in the investigator's laboratory, or in collaboration with a Russian research group. Analysis of the crosslinked peptides will show which regions of the subunits are in contact with RNA or DNA at particular stages of the transcription cycle. Mapping will also be facilitated by introduction of histidine tags into beta and beta', substitution of methionines at well defined, non-essential sites to facilitate mapping with cyanogen bromide, and cleavage of beta and beta' split into sub-domains prior to mapping.

描述：研究的主要焦点将集中在三个非常重要的方面 原核生物转录延伸的重要特征：(一) 加工性；(Ii)RNAP运动与核苷酸的关系 附加；(Iii)保真度和纠错。这项工作将基于一个 戈德法布博士最近提出了一种新的延伸率模型 构成了对先前持有的“尺寸虫”模型的修订。主 我们的想法是使用化学交联和蛋白质化学来定义 由不同功能组件或RNAP形成的触点，包括 前端DNA结合位点(DBS)、催化活性中心(AC)、 RNA-DNA杂交结合位点(HBS)，以及上游RNA产物结合位点 (苏格兰皇家银行)。此外，调查员建议解剖基本的 核苷酸加成反应进入易位前和易位后状态。 在过去的五年中，一些创新的技术被 主要是在Goldfarb实验室开发的，它提供了复杂的 用于研究结构-功能关系的工具 延长：(I)使用His标记的酶进行RNAP步行实验 螯合到镍-琼脂糖珠上，从而允许分离明确的 每一步都很复杂。(Ii)可将交联型探头并入 在5‘和3’末端或在转录本正文内的RNA，以及 在DNA模板上的任何位置。然后可以使用这些工具来演示 RNAP的哪些部分被标记的核苷酸接触，并且还 被用来证明新生的RNA-DNA的形成和范围 混血儿。(3)使用了衍生的可交联型利福平化合物 以确定RIF结合位点和RIF结合位点之间的关系 转录起始点。(4)Fe++诱导的卵裂有助于识别 酶中组成活性中心(AC)的部位。许多这样的 已经使用方法来表征活性中心，这似乎是 由来自测试版和测试版的不同区域的模块组成。 提议的实验的基本设计将使用特定的 放射性交联剂在特定位置被取代 新生的RNA或在DNA模板中，随后裂解交联体 具有几种不同氨基酸专一性切割的蛋白质亚单位 试剂。具有不同交联度或不同交联度的各种交联剂 在新生RNA中被放置在不同位置的能力将是 其中许多都是在研究人员的实验室中开发出来的， 或者与一家俄罗斯研究小组合作。三、分析了 交联肽将显示亚基的哪些区域相互接触 在转录周期的特定阶段与RNA或DNA结合。映射 还将通过将组氨酸标签引入测试版和 β‘，蛋氨酸在定义明确的非必需位点上的替代 便于用溴化氰作图，并切割β和β‘ 在映射之前拆分为子域。