REGULATION OF GENE EXPRESSION IN CHLAMYDIA

沙眼衣原体是一种主要病原体，具有不寻常的发展。 生命周期。这项建议的目的是研究其发病机制。 通过表征基因表达的调节对衣原体的影响 与细胞内生命周期的不同阶段有关 有机体。衣原体基因调控的研究受到阻碍 由于缺乏自然产生的或实验的基因方法 转移和大量培养生物的困难。 我们的方法是使用体外衣原体转录系统 描述监管流程的特征。这个转录系统将 分两条路发展。RNA聚合酶(RNAP)将在生物化学上 从具有转录活性的衣原体粗提物中提纯。RNAP 也将由单独过度表达的克隆亚基组装而成 在从大肠杆菌中提纯的。 启动子和DNA调控元件将由删除和 克隆的衣原体启动子区域的突变分析 启动子活性在体外系统中作为功能分析。推动者 结构也将通过足迹的物理分析来定义 通过营养衣原体因子与RNAP和启动子结合。 与DNA调控元件结合的反式作用因子将是 用酵母体内筛选或竞争亲和力鉴定 层析法。衣原体RNAP的特征是 组装体上个别亚基的缺失和点突变 全酶的作用。衣原体亚基的多肽片段 将用于定义In启动子的不同区域的作用 认可和核心约束。通过这种方式，衣原体的基本生物学 可以被探索，并可以获得对这一疾病的发病机制的见解 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.