Rho-dependent Termination within the trp t ‘ Terminator. II. Effects of Kinetic Competition and Rho Processivity

Abstract

Continuing our quantitative analysis of rho-dependent termination at the trp t ' terminator, we here present evidence that the position of rho-dependent terminators along the template is strongly regulated by the secondary structure of the nascent RNA transcript, and that the prerequisite for establishing an effective kinetic competition between elongation and rho-dependent RNA release at a particular termination position is an upstream rho hexamer properly bound to a rho loading site on the nascent transcript. As a consequence kinetic competition regulates termination efficiency at individual positions downstream of the rho loading site, but does not control the position of the termination zone. Conditions that favor the formation of stable secondary structure on the RNA shift the initial rho-dependent termination position downstream. These results are consistent with a model that states that the rho protein requires approximately 70-80 nucleotide residues of unstructured RNA to load onto the transcript and cause termination, and that stable 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 transcript elongation is important in determining termination efficiency at specific template positions, the process of loading of the rho hexamer 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 rate of transcript elongation, rho-dependent transcript termination is more directly dependent on the efficiency of rho loading, since the processivity of translocation of rho along the nascent transcript to "catch up with" the polymerase is much more limited under these conditions. A quantitative model for rho-dependent transcript termination is developed to account for all these interacting effects of rho on the efficiency of RNA release from actively transcribing elongation complexes.