REGULATION OF GENE EXPRESSION IN CHLAMYDIA

沙眼衣原体是一种主要的病原体， 生命周期 本提案的目的是研究发病机制 通过表征基因表达的调节来研究衣原体 与细胞内生命周期的不同阶段相关 有机体 衣原体基因调控的研究受到阻碍 由于缺乏自然发生的或实验性的方法， 转移和难以大量生长的有机体。 我们的方法是使用体外衣原体转录系统， 描述监管过程。 该转录系统将 以两种方式发展。 RNA聚合酶（RNAP）将被生物化学 从具有转录活性的粗衣原体提取物中纯化。 rnap 也将由单独过表达的克隆亚基组装 在纯化的E.杆菌 启动子和DNA调控元件将由缺失和缺失的序列定义。 克隆的假定衣原体启动子区域的突变分析，使用 在体外系统中的启动子活性作为功能测定。 启动子 还将通过足迹的物理分析来确定结构 与RNAP和启动子结合的营养衣原体O因子。 与DNA调控元件结合的反式作用因子将被 用酵母体内筛选或通过竞争性亲和力鉴定 层析 衣原体RNAP的特征在于 组装体上单个亚基的缺失和点突变， 全酶的功能。 衣原体亚单位的多肽片段 将用于定义In启动子不同区域的作用 识别和核心绑定。 这样衣原体的基本生物学 可以探索和见解可能获得的发病机制， 有机体

Chlamydia trachomatis is a major pathogen with an unusual development life-cycle. The objective of this proposal is to study the pathogenesis of Chlamydia by characterizing the regulation of gene expression associated with different stages in the intracellular life cycle of the organism. The study of gene regulation in Chlamydia has been hampered by the lack of naturally occurring or experimental methods for gene transfer and the difficulty in growing large quantities of the organism. Our approach is to use an in vitro chlamydial transcription system to characterize the regulatory processes. This transcription system will be developed in 2 ways. RNA polymerase (RNAP) will be biochemically purified from a transcriptionally-active crude chlamydial extract. RNAP will also be assembled from cloned subunits individually overexpressed in an purified from E. coli. Promoters and DNA regulatory elements will be defined by a deletional and mutational analysis of cloned putative chlamydial promoter regions, using promoter activity in the in vitro system as a functional assay. Promoter structure will also be defined by the physical assays of footprinting with RNAP and promoter binding by the vegetative chlamydial o factor. Trans-acting factors that bind to DNA regulatory elements will be identified with an in vivo screen in yeast or by competitive affinity chromatography. Chlamydial RNAP will be characterized by the effect of deletions and point mutations of individual subunits on the assembly and function of holoenzyme. Polypeptide fragments of the chlamydial subunit will be used to define the roles of different regions of in promoter recognition and core binding. In this way the basic biology of Chlamydia can be explored and insights may be gained into the pathogenesis of this organism.