Rho-dependent termination within the trp t' terminator. I. Effects of rho loading and template sequence

About one-half of the terminators of the Escherichia coli genome require transcription termination factor rho to function. Here we use the very "diffuse" trp t' terminator of E. coli to show that both template sequence and transcript secondary structure are involved in controlling the template positions and efficiencies of rho-dependent termination. Termination begins in the wild-type trp t' terminator sequence approximately 97 bps downstream of the promoter under our standard reaction conditions, and termination efficiencies for individual positions on three related templates have been determined in the form of quantitative patterns of rho-dependent RNA release. Comparison of these patterns shows that the rho-dependent termination efficiency at individual template positions depends primarily on the nucleotide sequence at and near the putative 3' end of the transcript, although these efficiencies can also be influenced by RNA sequence elements located further upstream. The amplitudes of the peaks of the RNA release patterns at specific template positions are controlled primarily by the effectiveness of the binding of the rho hexamer to the "rho loading site" of the transcript. Introduction of a stable element of secondary structure into the nascent RNA within the loading site both shifts the position of initial rho-dependent termination downstream and decreases the amplitudes of the peaks of the RNA release pattern at the corresponding sequences. These results and others are consistent with the view that rho-dependent terminators contain two essential components: (i) an upstream rho loading site on the RNA that is 70-80 nucleotide residues in length, essentially devoid of secondary structure, and which contains sufficient numbers of rC residues to activate the RNA-dependent ATPase of rho; and (ii) a downstream sequence within which termination actually occurs. In this study we use the trp t' terminator to characterize the involvement of each of these sequence components in detail in order to provide the parameters required to define a quantitative mechanistic model for the function of rho in transcript termination.