Figure 5.

Resumption of stalled complexes and Fe²⁺ cleavage indicate that EC20 complexes are not backtracked. (A) Complexes stalled at the gdh promoter at position +20 on a template with and without aryl azide derivatization at position +25 of the template strand (25T; lanes 2, 3 and 6, 7) were challenged after incubation for 5 min at 70°C with a complete set of NTPs (chase) for 2 min in the presence and absence of TFS as indicated on top of the figure. Lanes 1 and 10 contained RNA size markers. (B) Stalled immobilized EC20 were extensively washed in the absence of Mg²⁺, as indicated in ‘Materials and Methods’ section, and incubated for 20 min at 70°C. Reactions were then cooled to 20°C, and Fe²⁺ and DTT were added to initiate the cleavage reaction. The ∼18-nt Fe²⁺ cleavage product contains a 3′ phosphate causing as slightly higher electrophoretic mobility (39). The weak 21-nt RNA band is due to misincorporation at the G-residue of template DNA.The asteriks indicate non-specific bands.