STRUCTURE AND FUNCTION OF RNA POLYMERASE IN E COLI

RNA聚合酶(RNAP)是催化第一步的酶 在基因表达中--将遗传信息转录成信使 核糖核酸。因此，它是调节基因表达的因子的主要靶点 其基本功能和结构特征高度保守 从进化上讲。本项目的总体目标是了解 RNAP功能和调控的分子机制。与 X射线结晶学家戈德法布博士想要了解RNAP的结构 关于它与DNA、与核苷酸底物和与 它的RNA产品。单一的氨基酸替换将产生在 克隆的基因rpoB和rpoC，它们指定了RNAP的两个最大的亚基 全酶。这些突变将使用各种体外试验进行表征。 检测以确定哪些离散的酶活性受到影响。在……里面 此外，PI还建议开发一系列工程核酸模型 稳定地与RNAP结合形成类似于所形成的络合物的配体 在转录延伸过程中，这些配体将用于 与酶共结晶以完善结构模型和 确定酶在离散步骤中假定的构象差异 在转录延伸反应中。

RNA polymerase (RNAP) is the enzyme that catalyzes the first step in gene expression--the transcription of genetic information into messenger RNA. As such it is the principal target for factors regulating gene expression and its basic functions and structural features are highly conserved evolutionarily. The broad goal of this project is the understanding of the molecular mechanism of RNAP function and regulation. In collaboration with X-ray crystallographers, Dr. Goldfarb would like to understand RNAP structure in relation to its interactions with DNA, with nucleotide substrates, and with its RNA product. Single amino acid substitutions will be generated in the cloned genes, rpoB and rpoC, which specify the two largest subunits of RNAP holoenzyme. The mutations will be characterized using a variety of in vitro assays to determine which discrete activities of the enzyme are affected. In addition, the PI proposes to develop a series of engineered nucleic acid model ligands that bind stably to RNAP to form complexes analogous to those formed during transcription elongation; these ligands will be used for co-crystallization with the enzyme to refine the structural models and ascertain differences in conformations assumed by the enzyme at discrete steps in the transcription elongation reaction.