The crystal structure of the RNA-binding domain from Escherichia coli transcription termination factor Rho and implications for models of hexameric Rho

Transcription termination factor rho is an RNA- and ATP-dependent hexameric helicase found in most eubacterial species. The E. coli rho monomer consists of two domains, an RNA-binding domain (residues 1–130) and an ATPase domain (residues 131–419). The ATPase domain is homologous to the β subunit of F₁ ATPase. The RNA-binding domain shows no significant sequence similarity to any known proteins, but preliminary evidence suggested structural similarity to E. coli cold shock protein CspA. This dissertation describes the crystal structure determination of the RNA-binding domain (Rho130) from E. coli rho. Rho130 was cloned, expressed, and purified, and crystallization conditions were determined. X-ray crystallographic phases were determined through multi-wavelength anomalous dispersion (MAD) phasing upon seleno-methionine-containing crystals of Rho130. An initial model was built into a 1.8Å resolution map and the model was refined against 1.55Å resolution data. The final R-factor was 21.6% and R_free was 25.0%. The structure of Rho130 consists of an amino-terminal helical subdomain and a β-barrel subdomain. The β-barrel is a member of the oligonucleotide/oligosaccharide-binding (OB) fold and is similar to CspA. From an analysis of structural and biochemical data, an RNA-binding site on Rho130 has been identified. Finally, the β-barrel of Rho130 is also surprisingly similar to the amino-terminal β-barrel of F₁ ATPase, extending the applicability of F₁ ATPase as a structural model for hexameric rho.