RNA BIOSYNTHESIS IN ESCHERICHIA COLI

DNA依赖核糖核酸的结构-功能关系研究 提出了大肠杆菌的聚合酶(RNAP)，它将使用一种 蛋白质化学和酶学相结合的方法。这个 本研究的最终目的是了解RNAP的基本功能 和分子细节的调控，以及揭示潜在的 结构性决定因素。 本课题研究的重点是三元转录的拓扑结构。 当它沿着DNA传播时是复杂的。我们将测试该模型 复合体的运动是以“尺寸虫”的方式进行的， 酶以几个核苷酸的幅度压缩和伸展。 对RNA聚合酶繁殖机制的理解将提供 将这种酶设计为多功能分子机器的线索 它是调控基因表达的因子的靶标。 拟议的研究将重点放在三元的RNA成分上 很复杂。将使用三种主要方法。首先，我们打算 使用结合到指定位置的可交联亲和探针 转录本将RNA固定在复合体中，然后研究这样的复合体 酶促作用。正常核糖核酸链延伸的障碍 将有助于理解RNAP的机制 传播。 第二种方法使用了grea蛋白，这是一种新的转录裂解 我们发现的因子，作为三元RNA拓扑的探针 很复杂。Grea切割了在附近暴露的新生文本 酶的催化中心。从卵裂模式的变化看 发生在复杂的传播过程中，我们希望了解 酶运动的机制。 最后，我们建议确定与rna接触的氨基酸。 在三元复合体中，通过测定DNA-RNA杂交区的长度 用结合到转录本中的探针进行亲和标记 然后用蛋白质和核酸的方法绘制加合物的图谱 化学反应。

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.