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

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Abstract
Continuing our quantitative analysis of rho-dependent termination at the trp tterminator, 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 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. In the preceding paper (1) we showed that transcript termination under conditions of high overall rho-dependent termination efficiency can begin within the previously defined rho "loading site" of the trp tterminator even before the putatively essential rutB sequence has been transcribed. We also showed that the presence of RNA secondary struc- ture within the actual site of rho loading shifts the beginning of the rho-dependent termination zone downstream along the template that contains a trp tterminator, and that this effect can be relieved by destabilizing the secondary structure of the RNA by replacing guanosine residues in the transcript with inosine residues. This replacement resulted in no changes in the rate of synthesis by the transcription complex at the new termination sites, demonstrating that the presence of elements of RNA secondary structure within the actual rho loading site is primarily responsible for establishing the upstream limit of the zone within which rho-dependent termination can occur, and that the template position of this limit is independent of the rate of transcript elongation. Here we extend these observations by testing in detail the effects of the RNA secondary structure within the actual rho loading zone on: (i) the position of the upstream edge of the termination zone along the template; and (ii) the efficiency of rho-dependent termination at individual tem- plate positions within the termination zone. We show that decreasing the rate of transcript elongation does not change the position of the termination zone along the template, but does increase termination efficiency at individual termination positions. We further show that high concentrations of K+ or Mg2+ ions, which serve both to destabilize the binding of rho to the nascent RNA and to induce additional secondary structure in the transcript, do not increase the rate of transcript elongation. However, these increased cation concentrations do shift the termination zone downstream and decrease termination efficiency, and these salt concentration effects are only partially compensated by major decreases in the elongation rate of the transcription complex. Finally the results presented in this paper and the companion paper (1), together with the structural and mechanistic findings of others, are used to formulate a quantitative model for the efficiency of rho-dependent transcript termination as a function of template position, rho loading, and translocation processivity, and the kinetic competition between the rates of rho-dependent termination and transcript elongation.