Mutagenesis of region 4 of sigma 28 from Chlamydia trachomatis defines determinants for protein-protein and protein-DNA interactions.

Transcription factor sigma(28) in Chlamydia trachomatis (sigma(28)(Ct)) plays a role in the regulation of genes that are important for late-stage morphological differentiation. In vitro mutational and genetic screening in Salmonella enterica serovar Typhimurium was performed in order to identify mutants with mutations in region 4 of sigma(28)(Ct) that were defective in sigma(28)-specific transcription. Specially, the previously undefined but important interactions between sigma(28)(Ct) region 4 and the flap domain of the RNA polymerase beta subunit (beta-flap) or the -35 element of the chlamydial hctB promoter were examined. Our results indicate that amino acid residues E206, Y214, and E222 of sigma(28)(Ct) contribute to an interaction with the beta-flap when sigma(28)(Ct) associates with the core RNA polymerase. These residues function in contacts with the beta-flap similarly to their counterpart residues in Escherichia coli sigma(70). Conversely, residue Q236 of sigma(28)(Ct) directly binds the chlamydial hctB -35 element. The conserved counterpart residue in E. coli sigma(70) has not been reported to interact with the -35 element of the sigma(70) promoter. Observed functional disparity between sigma(28)(Ct) and sigma(70) region 4 is consistent with their divergent properties in promoter recognition. This work provides new insight into understanding the molecular basis of gene regulation controlled by sigma(28)(Ct) in C. trachomatis.