Chlamydia trachomatis is the leading cause of sexually transmitted diseases in this country and a major cause of blindness in third world countries. This obligate intracellular parasite has a unique intracellular life cycle that is central to its pathogenesis. Remarkably little is known about regulation of this life cycle, in part due to the difficulty in growing large quantities of the organism in tissue culture, the lack of chlamydial mutants, and the inability to genetically manipulate these organisms. This grant proposes to dissect the regulation of transcription initiation during the chlamydial developmental life cycle using in vitro transcription. We will define the cis-acting and trans-acting factors necessary and sufficient to direct transcription in an in vitro system, using constitutively expressed and developmentally regulated promoters that we have previously isolated and characterized. Understanding the regulation of the life cycle will yield insights into chlamydial disease pathogenesis as well as insights into gene regulation. Specific Aim 1: We will reconstitute chlamydial transcription in vitro using two approaches. First, we will prepare a transcriptionally-active chlamydial cell-free extract from reticulate bodies. Second, we will reconstitute pure chlamydial RNA polymerase (RNAP) from cloned subunits individually overexpressed in and purified from E. coli. Specific Aim 2: We will define promoters and other cis-acting elements controlling transcription of cloned constitutive and regulated genes using a functional assay and a physical assay. We will identify promoter sequences by testing the effect upon transcription of deletions and point mutations in cloned chlamydial promoters using both the crude extracts and the reconstituted purified chlamydial RNAP holoenzyme. We will define contacts of RNAP with the wild type and selected mutant promoters by footprint analysis. We will compare the results of these two assays using both constitutive promoters and regulated promoters as templates. Specific Aim 3: We will identify trans- acting factors that are involved in regulating gene expression. We will clone chlamydial proteins that demonstrate specific binding to DNA regulatory sequences using an in vivo screen in S. cerevisiae or E. coli. These DNA-binding proteins will be tested for their regulatory effect on transcription in the crude and reconstituted in vitro transcription systems. Specific Aim 4. We will carry out a genetic analysis of the regulation of the chlamydial groE and dnaK heat shock operons using B. subtilis as a surrogate genetic host. We will analyze the promoter and other cis-acting regulatory sequences controlling the transcription of the heat shock operons groE and dnaK using B. subtilis as a surrogate host. We will clone and characterize the chlamydial regulatory proteins that regulate transcription from the groE and dnaK heat shock operons by complementation of B. subtilis mutants. From these studies may emerge new therapeutic approaches for the treatment and prevention of chlamydial infections.