RNAs that are not protected by ribosomes or antitermination complexes are prematurely terminated by Rho, a hexameric ATP-dependent RNA helicase. As a genome sentinel, Rho ensures that only “worthy” RNAs are made: Rho blocks synthesis of antisense and xenogeneic RNAs, as well as mRNAs that contain early stop codons. The textbook model posits that Rho binds to the nascent RNA and uses ATP hydrolysis to translocase on the RNA and to pull it out of RNAP, thus terminating transcription. Our recent results contradict this view and support a model in which Rho travels with RNAP and induces dramatic conformational changes that allosterically inactivate the elongation complex, causing RNAP to lose its grip on the DNA and dislodging the 3’ end of the RNA from the active site, while never engaging Rho ATPase motor.
A model in which Rho rides along with elongating RNAP explains how Rho selectively targets and surveils the nascent RNAs, but raises a key question: how is indiscriminate termination avoided during translational stress or even slow growth? In the lab we feed bacteria well, but in their native habitats resources are scarce and long periods of inactivity are common, implying the existence of mechanisms that prevent Rho from going rogue. Since Rho cellular levels are constant, cellular proteins or small molecules may sequester Rho in an inactive state, blocking its interactions with the elongation complex.
In collaboration with Markus Wahl and Peter Freddolino, we are working on two mechanisms of Rho control. First, we selected partial-loss-of-function rho mutants that promote formation of long helical filaments that resemble those of Rho homolog RecA. Filamentation is a widespread mechanism of adaptation to stress in all domains of life, and we hypothesize that wild-type Rho may also form hibernating filaments in response to yet-unknown cellular cues. Second, we are elucidating the molecular mechanisms of small phage and bacterial proteins which have been shown to inhibit Rho activity when overexpressed.