RNA BIOSYNTHESIS IN ESCHERICHIA COLI

大肠杆菌中的 RNA 生物合成

• 批准号: 2468901
• 负责人: Alexander Goldfarb
• 金额: $ 30.01万
• 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/2468901
• 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：研究的主要重点将放在三个非常 原核转录延伸的重要特征：（i） 持续合成能力;（ii）RNAP运动和核苷酸之间的关系 加法;（iii）保真度和纠错。 这项工作将基于 Goldfarb博士最近提出的一种新的伸长模型， 构成对先前持有的“尺蠖”模型的修订。 主要 想法将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杂交结合位点（HBS）和上游RNA产物结合位点 （RBS）. 此外，研究人员建议剖析基本的 将核苷酸加成反应转化为易位前和易位后状态。 在过去的五年里，一些创新技术已经被 主要是在Goldfarb实验室开发的， 研究结构-功能关系的工具， 延伸：（i）RNAP步行实验用His标记的酶进行 螯合到Ni-琼脂糖珠，从而允许分离限定的 在每一步的配合。 (ii)交联探针可以并入到 在转录物的5'和3'末端或体内的RNA，以及 DNA模板上的任何位置。 这些可以用来证明 RNAP的哪些部分与标记的核苷酸接触，并且还具有 已经被用来证明新生RNA-DNA的形成和程度， 杂种 (iii)衍生的可交联利福平化合物已被用于 为了鉴定RIF结合位点和 转录起始位置。 (iv)Fe++诱导的分裂有助于确定 酶中的活性中心（AC）。 其中一些 方法已被用来表征活性中心，这似乎是 由来自β和β '的不同区域的模块组成。 拟议实验的基本设计将使用特定的 放射性交联剂在特定位置取代， 新生RNA或DNA模板中，然后裂解交联的 具有几种不同氨基酸特异性切割的蛋白质亚单位 试剂 具有不同交联半径的各种交联剂，或具有 在新生RNA中被放置在不同位置的能力， 其中许多是在研究人员的实验室中开发的， 或与俄罗斯研究小组合作。 分析 交联的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。 映射 也将通过将组氨酸标签引入β和 β '，在明确定义的非必需位点取代甲硫氨酸， 便于用溴化氰作图，以及β和β '的切割 在映射之前分成子域。