RNA BIOSYNTHESIS IN ESCHERICHIA COLI

大肠杆菌中的 RNA 生物合成

• 批准号: 3308590
• 负责人: Alexander Goldfarb
• 金额: $ 25.97万
• 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 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308590
• 关键词: DNA directed RNA polymerase; Escherichia coli; RNA; affinity labeling; autoradiography; bacterial genetics; crosslink; enzyme activity; gene expression; genetic promoter element; high performance liquid chromatography; immobilized enzymes; nucleic acid biosynthesis; nucleic acid hybridization; nucleic acid probes; protein structure function; radiotracer; transcription factor

A study of structure-function relationships in DNA-dependent RNA polymerase (RNAP) of Escherichia coli is proposed which will use a combination of protein-chemical and enzymological approaches. The ultimate goal of this study is the understanding of RNAP basic function and regulation in molecular detail, and the uncovering of the underlying structural determinants. The emphasis of this project is on topology of the ternary transcription complex as it propagates along DNA. We will test the model that the movement of the complex occurs in the "inchworm" fashion whereby the enzyme compresses and extends with the amplitude of several nucleotides. Understanding of the mechanism of RNA polymerase propagation will provide clues to the design of this enzyme as a multifunctional molecular machine which serves as the target to factors that regulate gene expression. The proposed study will be focused on the RNA component of the ternary complex. Three principal approaches will be used. First we intend to use crosslinkable affinity probes incorporated into defined positions in the transcript to fix RNA in the complex, and then to study such complex enzymatically. The impediment to the normal RNA chain extension resulting from the crosslink will help understand the mechanism of RNAP propagation. The second approach employs the GreA protein, a novel transcript cleavage factor that we discovered, as a probe of RNA topology in the ternary complex. GreA cleaves the nascent transcript exposed in the vicinity of the enzyme's catalytic center. From the changes of the cleavage pattern that take place during complex propagation we expect to learn about the mechanism of the enzyme movement. Finally, we propose to identify amino acids involved in contacts with RNA in the ternary complex, and the length of DNA-RNA hybrid region by means of affinity labeling with probes incorporated into the transcript followed by mapping the adducts using methods of protein and nucleic acid chemistry.

DNA依赖性RNA结构与功能关系的研究 提出了大肠杆菌聚合酶（RNAP），该酶将使用 蛋白质化学和酶学方法的结合。 这 本研究的最终目标是了解RNAP的基本功能 分子细节的调控，以及潜在的揭示 结构性决定因素。 该项目的重点是三元转录的拓扑结构 当它沿着 DNA 传播时会变得复杂。 我们将测试该模型 复合体的运动以“尺蠖”的方式发生，由此 酶以几个核苷酸的幅度压缩和延伸。 了解 RNA 聚合酶传播的机制将提供 这种酶作为多功能分子机器的设计线索 它作为调节基因表达的因子的靶标。 拟议的研究将集中于三元RNA的RNA成分 复杂的。 将使用三种主要方法。 首先我们打算 使用掺入到确定位置的可交联亲和探针 转录物将RNA固定在复合物中，然后研究这种复合物 酶促地。 正常RNA链延伸的障碍 交联产生的结果将有助于理解 RNAP 的机制 传播。 第二种方法采用 GreA 蛋白，一种新型转录本切割 我们发现的因子，作为三元RNA拓扑结构的探针 复杂的。 GreA 切割暴露在附近的新生转录物 酶的催化中心。 从解理图案的变化来看 在复杂的传播过程中发生的事情我们希望了解 酶运动的机制。 最后，我们建议鉴定与 RNA 接触有关的氨基酸 在三元复合物中，DNA-RNA杂合区的长度通过 用掺入转录本的探针进行亲和标记 然后使用蛋白质和核酸的方法绘制加合物图谱 化学。