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：研究的主要重点将集中在三个方面： 原核生物转录延伸的重要特征：（i） 持续性； (ii)RNAP运动与核苷酸之间的关系 添加; (iii) 保真度和纠错。 这项工作将基于 Goldfarb 博士最近提出了新的伸长模型， 构成对先前持有的“inchworm”模型的修订。 主要 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 形成的接触，包括 前端DNA结合位点(DBS)、催化活性中心(AC)、 RNA-DNA 混合结合位点 (HBS) 和上游 RNA 产物结合位点 （苏格兰皇家银行）。 此外，研究者建议剖析基本的 核苷酸加成反应进入易位前和易位后状态。 五年来，多项创新技术得到应用 主要在 Goldfarb 实验室开发，提供先进的 研究结构与功能关系的工具 伸长：(i) RNAP 步行实验是用 His 标记的酶进行的 与镍琼脂糖珠螯合，从而可以分离指定的 每一步都有复合物。 (ii) 交联探针可以并入 5' 和 3' 末端或转录本体内的 RNA，以及 DNA 模板上的任意位置。 然后可以使用这些来演示 RNAP 的哪些部分与标记的核苷酸接触，并且还具有 用于证明新生 RNA-DNA 的形成和范围 杂交种。 (iii) 使用了衍生的可交联利福平化合物 确定 rif 结合位点和 转录起始位点。 (iv) Fe++ 诱导的裂解有助于识别 酶中构成活性中心（AC）的位点。 其中一些 方法已被用来表征活性中心，这似乎 由来自 beta 和 beta' 不同区域的模块组成。 所提出的实验的基本设计将是使用特定的 放射性交联剂在特定位置被取代 新生 RNA 或 DNA 模板中，然后切割交联的 具有几种不同氨基酸特异性切割中的任何一种的蛋白质亚基 试剂。 具有不同交联半径的各种交联剂，或具有 被放置在新生RNA中不同位置的能力，将是 其中许多是在研究者的实验室中开发的， 或与俄罗斯研究小组合作。 分析 交联肽将显示亚基的哪些区域是接触的 与RNA或DNA在转录周期的特定阶段。 测绘 将组氨酸标签引入 beta 和 beta'，在明确定义的非必需位点替换甲硫氨酸 促进溴化氰作图，以及 β 和 β' 的裂解 在映射之前分割成子域。