H-078. A Novel Chlamydial Protein Binds to the β Subunit of RNA Polymerase

X. Rao1, Z. Hua1, P. Deighan2, Y. Liang1, A. Hochschild2, L. Shen1;
1Boston Univ. Sch. of Med., Boston, MA, 2Harvard Med. Sch., Boston, MA.

The developmental gene expression in Chlamydia is mainly regulated at the level of transcription that is mediated by RNA polymerase (RNAP) holoenzyme with composition α2ββ’ω (core) and σ subunit. Using a two-hybrid based genetic screening, we identified the chlamydial CT663 interacting with the flap domain of β subunit of RNAP (β-flap). CT663 protein was associated with both E. coli and chlamydial β subunits expressed in E. coli using a Co-IP assay. Purified GST-fused CT663 protein was able to pulldown β subunits from fractions of E. coli cellular extraction. In addition, purified His-tagged CT663 protein complexes with the core as well as the σ70 holoenzyme in a native protein gel. We find that CT663 protein interacts with β subunit at the tip helix structure of β-flap. Furthermore, we identified the amino acid residues 901L, 905I and 906F of E. coli β subunit as the determinants in the E. coli β-flap that are essential for CT663 protein interaction with an alanine substitution library screening. The corresponding residues in chlamydial β (841L, 845I and 846F) are similarly important for β-flap/CT663 interaction. Previous work has shown that the same amino acids in β-flap tip helix are contacted by the major sigma factors as well. We propose that the CT 663 protein may target at the intersubunit interaction within the RNAP holoenzyme. Further experiments are on the way to test the hypothesis.