Poster abstracts
Poster number 17 submitted by Mark Finazzo
Rof: an oddly coupled inhibitor of Rho
Mark Finazzo (The Ohio State University), Nelly Said (Freie Universitt Berlin), Bing Wang (The Ohio State University), Markus C. Wahl (Freie Universitt Berlin), Irina Artsimovitch (The Ohio State University)
Abstract:
Bacterial Rho is a hexameric RNA helicase that terminates synthesis of useless or harmful RNAs, while actively translated messages are protected by the ribosomes. When translation is limiting, the cell may need to reduce Rho activity. Two Escherichia coli inhibitors of Rho are known: SrkA (aka YihE), a Ser-Thr kinase induced by periplasmic stress, and Rof, a structural homolog of Hfq. Using cryoEM and functional analyses, we recently elucidated the mechanism of Rof. Rof binds at the interface of two Rho protomers, locking Rho in an inactive open-ring conformation and blocking its binding to RNA and RNA polymerase, thereby inhibiting termination. Biochemical and genetic assays validated the interacting residues identified in a cryoEM structure. A growth defect was observed when wild-type Rof was overexpressed in the cell, whereas substitutions of key Rof residues abolished Rof toxicity in vivo and inhibition of Rho-dependent termination in vitro. The cellular conditions that trigger Rof activity are yet unknown but, intriguingly, E. coli rof is encoded within a stress-associated locus and overlaps with an upstream gene neighbor of unknown function, yaeP. Within the overlap, there are two start codons in rof’s ORF, yielding two isoforms differing in length by only two residues. Modifying yaeP ORF abolishes the expression of Rof, suggesting that their translation is obligatory coupled, a potential mode of regulation of Rof activity that we are actively investigating.
Keywords: Rho, transcription, termination