REGULATION OF GENE EXPRESSION IN CHLAMYDIA

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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.