Three kinetically distinct but compatible routes of Rho-dependent termination uncovered by single-molecule transcription imaging.

Abstract

When gene transcription for RNA synthesis terminates in a factor-dependent manner, Rho is a general termination factor in bacteria, but its mechanism has been elusive in many aspects. Various models have been proposed for the pre-terminational interaction of Rho with RNA polymerase (RNAP), for the terminational release of RNA transcript, and for the post-terminational outcome. We developed several single-molecule fluorescence assays in order to monitor those three steps of termination mediated by Escherichia coli Rho. We find that the different mechanisms previously proposed for each step co-exist compatibly together in any single terminator but three kinetically distinct routes take place with each terminator. Rho mostly binds nascent RNA first at a rut site, translocates on RNA and catches up with the paused RNAP at the termination site. The fastest route is that this catch-up Rho first mediates RNA shearing for RNA-only release and consequently one-dimensional recycling of the DNA-bound RNAP. The catch-up Rho later mediates RNAP displacement in the next but the most frequent route to decompose RNAP·DNA·RNA complexes in a step for three-dimensional recycling of the dissociated RNAP. Contrastingly, a minor fraction of Rho pre-binds RNAP prior to the terminational pausing site and stands by for an incipient RNA rut site. This stand-by Rho lastly mediates only RNAP displacement for the decomposing outcome in the slowest route, despite interacting with RNAP earlier than the catch-up Rho. Furthermore, we uncover that transcriptional pause extension benefits only the stand-by Rho's termination, and the essential pause does not need to be long for the catch-up Rho's terminations The riboswitch-associated termination is controlled mainly by modulation of the stand-by. Thus, the three compatible routes of Rho-dependent termination not only operate on distinct timescales but also play different regulatory roles.