RHO-DEPENDENT TERMINATION WITHIN THE TRP T'-TERMINATOR - II - EFFECTSOF KINETIC COMPETITION AND RHO-PROCESSIVITY

Continuing our quantitative analysis of rho-dependent termination at t he trp t' terminator, we here present evidence that the position of rh o-dependent terminators along the template is strongly regulated by th e secondary structure of the nascent RNA transcript, and that the prer equisite for establishing an effective kinetic competition between elo ngation and rho-dependent RNA release at a particular termination posi tion is an upstream rho hexamer properly bound to a rho loading site o n the nascent transcript. As a consequence kinetic competition regulat es termination efficiency at individual positions downstream of the rh o loading site, but does not control the position of the termination z one. Conditions that favor the formation of stable secondary structure on the RNA shift the initial rho-dependent termination position downs tream. These results are consistent with a model that states that the rho protein requires similar to 70-80 nucleotide residues of unstructu red RNA to load onto the transcript and cause termination, and that st able RNA secondary structures are effectively ''looped out'' to avoid interaction with rho, meaning that more RNA must be synthesized before rho-dependent termination can begin. Thus, although the rate of trans cript elongation is important in determining termination efficiency at specific template positions, the process of loading of the rho hexame r onto the nascent transcript plays an overriding role in determining the template positions of rho-dependent terminators. We also show that at high salt concentrations, which have virtually no effect on the ra te of transcript elongation, rho-dependent transcript termination is m ore directly dependent on the efficiency of rho loading, since the pro cessivity of translocation of rho along the nascent transcript to ''ca tch up with'' the polymerase is much more limited under these conditio ns. A quantitative model for rho-dependent transcript termination is d eveloped to account for all these interacting effects of rho on the ef ficiency of RNA release from actively transcribing elongation complexe s.