H-079. Analysis of DNA-Protein Interaction Mediated by Carboxyl-Terminal Residues in Chlamydial Sigma 28

Z. Hua, X. Rao, Y. Liang, L. Shen;
Boston Univ. Med. Sch., Boston, MA.

Chlamydial σ²⁸ (σ²⁸_Ct) RNA polymerase holoenzyme (RNAP) regulates transcription of genes important for the late-stage differentiation in Chlamydia. To test the importance of residues within σ²⁸_Ct region 4 (σ²⁸_CtR4) function, we investigated how σ²⁸_CtR4 mutants interact with the -35 element of σ²⁸_Ct-dependent promoter (hctB)_. In vitro mutation and screening in Salmonella typhimurium were used to identify mutants in σ²⁸_CtR4 that affect σ²⁸_Ct-specific transcription. The RNAP containing corresponding mutant σ²⁸_Ct was tested for their ability to transcribe at chlamydial hctB promoter in Salmonella and in vitro. Our results demonstrated that the substitutions in σ²⁸_Ct R4 (E206G, M210T, Y214C, E222G and Q236L) reduced in vivo transcription from hctB. In vitro the substitutions E206G, Y214C, E222G and Q236L decreased transcription initiating from hctB as well. Furthermore, a one-hybrid assay was employed to examine the effect of σ²⁸_CtR4 mutants on its interaction with the chlamydial hctB -35 element (hctB-35, TAAAGTTT). This assay is based on a reporter strain harboring specialized ectopic hctB-35 upstream of plac::lacZ on its F’ episome and a plasmid-encoded chimera protein with N-terminal domain of α subunit fused to the σ²⁸_CtR4. The optimal location of ectopic hctB-35 recognized by the σ moiety of α-σ²⁸_Ct chimera is at -45.5 bp upstream from the transcription start point. This result agrees with the reported optimal location of ectopic lac-35 recognized by α-σ⁷⁰_Ct chimera. Mutation of hctB-35 “gAAAGTTT” weakens hctB-35/α-σ²⁸_Ct interaction. The substitution Q236L failed to stimulate transcription from the artificial promoter placCons-35(hctB), likely to disrupt the ability of σ²⁸R4 to interact with hctB-35. All these mutations in σ²⁸_CtR4 did not cross-interact with the σ⁷⁰ specific -35 element (TTGACA). Our data provides evidence that the side-chain of residue Q236 might be involved in the interaction between σ²⁸_CtR4 and hctB -35. Further studies are needed to test the potential mechanisms that other σ²⁸_CtR4 mutants may involve in the transcription process.