Poster abstracts
Poster number 55 submitted by Nicholas Sunday
Investigating the Role of the Connector Region in E. coli Termination Factor Rho
Nicholas Sunday (Department of Microbiology), Max Gilliland (Department of Microbiology), Marcos Sotomayor (Department of Chemistry & Biochemistry), Irina Artsimovitch (Department of Microbiology)
Abstract:
Transcription termination factor Rho is an ancient hexameric protein that is present in most bacteria. Rho monomer is composed of two domains separated by a flexible connector. The N-terminal domain (NTD) contains a primary RNA-binding site, the C-terminal domain - the secondary RNA-binding site, ATPase and helicase modules. In a textbook model, Rho-NTD binds to a pyrimidine-rich rut site in the RNA, triggering a switch from an open state to an active, closed-ring state in which the nascent RNA is trapped within Rho-CTD. ATP turnover then promotes Rho translocation along the nascent RNA until Rho encounters and releases RNA polymerase. This mechanism is supported by in vitro studies on model substrates but fails to account for how Rho works on myriad RNAs that lack rut sequences, such as xenogenes, antisense transcripts, and R-loops. Using a selection for Escherichia coli mutants defective in termination at suboptimal sites, we identified novel substitutions in the Rho connector that are predicted to reduce its flexibility. We and others also showed that the transcription elongation factor NusG cooperates with Rho to induce termination at non-canonical sites. We hypothesize that the connector mediates allosteric communications between the primary and secondary RNA-binding sites that are necessary for ring closure. We plan to combine in silico analyses of Rho dynamics with in vitro and in vivo assays of Rho-dependent termination to test if the connector flexibility facilitates the formation of the catalytically-active state of Rho and is particularly critical at suboptimal sites.
Keywords: Rho, Termination, Regulation