STRUCTURAL AND FUNCTIONAL STUDIES OF AN ATYPICAL OMPR/PHOB TRANSCRIPTIONAL REGULATOR, CHXR, FROM CHLAMYDIA TRACHOMATIS

Abstract

Chlamydia infections have an immense impact on public health and are associated with diverse disease manifestations including atherosclerosis, blindness, and sterility. The chlamydial developmental cycle is intrinsically linked with the ability of the organism to cause disease. The mechanisms that regulate the developmental cycle are poorly understood; however, transcription appears to play a governing role. An OmpR/PhoB subfamily response regulator termed ChxR exhibits expression patterns that indicate an important role during the developmental cycle. Previously, ChxR was demonstrated to interact with its own promoter and facilitate the transcriptional activation of the chxR gene. To begin to understand the functional role of ChxR, I identified the DNA sequence recognized by ChxR to identify its gene targets. Primarily using gel mobility shift assays, I determined that ChxR interacts with, and has differential affinity for six binding sites in the chxR promoter region. Using the DNA sequences from these binding sites, I elucidated the ChxR cis-acting recognition sequence. Additionally, I was interested in elucidating the ChxR mechanism of transcriptional activation. Usually as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Dimer formation facilitates an interaction of the effector domain interaction with DNA and transcriptional machinery to regulate transcription. ChxR is an atypical OmpR/PhoB response regulator because it is active in the absence of phosphorylation. We hypothesized that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR protein are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. Using biochemical techniques, I demonstrated that ChxR forms homodimers through the receiver domain and the effector domain interacts with DNA similar to phosphorylation-induced and transcriptionally active OmpR/PhoB response regulators. Additionally, the structures of the two domains were solved to direct functional studies to identify the residues important in homodimer formation, interaction with DNA, and interaction with RNA polymerase machinery. Both structures had unique features that are not found in other OmpR/PhoB subfamily members. The combination of these results suggests that ChxR is a member of the OmpR/PhoB subfamily, although many of the characteristics of the subfamily are not shared in ChxR.