RNA BIOSYNTHESIS IN ESCHERICHIA COLI

依赖DNA的RNA结构与功能关系的研究 提出了大肠杆菌的RNAP聚合酶，其将使用 结合蛋白质化学和酶学方法。 的 本研究的最终目的是了解RNAP的基本功能 和调节的分子细节，以及揭示潜在的 结构决定因素 本研究的重点是三重转录的拓扑结构 当它沿着沿着DNA传播时， 我们将测试模型， 复合体的运动以“尺蠖”的方式发生， 酶以几个核苷酸的幅度压缩和伸展。 了解RNA聚合酶增殖的机制将提供 设计这种酶作为多功能分子机器的线索 其作为调节基因表达的因子的靶标。 拟议的研究将集中在RNA组分的三元 复杂. 将采用三种主要方法。 首先，我们打算 使用可交联的亲和探针， 转录本将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 这将有助于理解RNAP的作用机理 传播 第二种方法采用GreA蛋白，一种新的转录物切割 我们发现，作为RNA拓扑结构的探针， 复杂. GreA切割暴露在 酶的催化中心 从卵裂模式的变化来看 在复杂的传播过程中发生，我们希望了解 酶的运动机制。 最后，我们建议确定与RNA接触的氨基酸 在三元复合物中，和DNA-RNA杂交区的长度， 将探针结合到转录物中进行亲和标记 然后使用蛋白质和核酸的方法绘制加合物 化学.

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.