RNA BIOSYNTHESIS IN ESCHERICHIA COLI

大肠杆菌中的 RNA 生物合成

基本信息

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
项目类别：
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

项目摘要

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：研究的主要重点将是三个非常核转录伸长的重要特征：（i）加工性；（ii）RNAP运动与核苷酸之间的关系添加; （iii）保真度和误差校正。这项工作将基于 Goldfarb博士最近提出了有关伸长率的新模型，该模型构成了先前持有的“ Inch虫”模型的修订。主想法是使用化学交联和蛋白质化学定义由不同功能组件或RNAP建立的触点，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）和上游RNA产物结合位点（RB）。此外，研究人员建议剖析基本核苷酸添加反应到转升前和后置状态。在过去的五年中，许多创新技术开发，主要是在Goldfarb实验室中开发的，该实验室提供了精致的调查结构功能关系的工具伸长率：（i）用他的标签酶进行RNAP步行实验螯合到Ni-agarose珠，从而允许隔离定义每个步骤的复合物。（ii）可以将交联探针纳入在5'和3'末端或成绩单的正文内的RNA以及在DNA模板上的任何位置。然后可以使用这些证明标记的核苷酸与RNAP的哪一部分联系在一起，也有用于证明新生RNA-DNA的形成和程度杂交种。（iii）已使用了衍生化的可连接利福平化合物确定RIF结合位点与转录启动。（iv）Fe ++诱导的裂解有助于确定组成活性中心（AC）的酶中的位置。其中一些方法已被用来表征活跃中心，似乎由来自Beta和Beta的不同区域的模块组成。拟议实验的基本设计将是使用特定放射性交联剂在特定位置取代新生的RNA或DNA模板中，然后切开交联蛋白质亚基，几个不同的氨基酸特异性裂解中的任何一个试剂。具有不同的交联半径或与将其放置在新生RNA的不同位置的能力将是雇用了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接触的亚基的哪些区域在转录周期的特定阶段中带有RNA或DNA。映射还将通过将组氨酸标签引入beta和 beta'，在定义良好的，不必要的地点替代蛋白促进用氰基溴的映射，并裂解β和β' 在映射之前分为子域。