Extended Data Fig. 2: Scaffold assay for Rho-dependent termination: additional data.

(a) Top, synthetic nucleic-acid scaffolds containing complementary nontemplate and template DNA strands (colours as in Fig. 1b; Extended Data Fig. 1). Bottom, RNA-extension data assessing Rho-dependent termination in complexes having λtR1 rut as PBS ligand (odd-numbered lanes) or dC75 as PBS ligand (even-numbered lanes) on scaffolds where the U-rich-RNA-segment length, n, is 4, 5, 6, 7, 8 or 9 codons (first, second, third, fourth, fifth, and sixth subpanels). Red asterisks, RNA products that exhibit increased levels compared to control reactions without PBS ligand; open circles, RNA products that exhibit decreased levels relative to control reactions without PBS ligand. Termination is detected by an increase in unextended and partly extended RNA products (lower and central bands), and a decrease in fully extended RNA products (top bands). Assays were performed twice with consistent results. (b) As in a, except that synthetic nucleic-acid scaffolds contain noncomplementary nontemplate and template DNA strands. The PBS ligand λtR1 rut supports efficient and immediate termination under these conditions; with λtR1 rut, approximately 100% of RNA synthesis ceases before RNA extension (lane 1 of each gel panel). The PBS ligand dC75 supports efficient but less immediate termination under these conditions; with dC75, approximately 30% of RNA synthesis ceases before RNA extension, and approximately 70% of RNA synthesis ceases only after RNA extension by 1 nt or 2 nt (lane 2 of each gel panel). The results indicate that both λtR1 rut and dC75 are effective PBS ligands and that λtR1 rut is a more effective PBS ligand than dC75 (see Extended Data Fig. 1). All six tested U-rich-RNA-segment lengths (n = 4, 5, 6, 7, 8, and 9 codons) support termination. In the experiments of a, the six tested U-rich-RNA-segment lengths support termination comparably efficiently. In the experiments of b, shorter lengths (n = 4, 5, and 6 codons) support termination more efficiently than longer lengths (n = 7, 8, and 9 codons).