Figure 2.

Covalent trapping of the extended ribozyme-substrate complex with T4 RNA ligase. Substrate cleavage reactions were carried out either in the absence (•) or in the presence (○) of T4 RNA ligase (see Materials and Methods). Time courses were fitted to double- or single-exponential equations, respectively (see Materials and Methods). Amplitudes (A₁ and A₂) and rates (r₁ and r₂) for the biphasic reaction in the absence of RNA ligase were A₁ = 0.71, r₁ = 0.13 min⁻¹; A₂ = 0.19, r₂ = 0.01 min⁻¹. Parameters for the monophasic reaction in the presence of RNA ligase were A = 0.73, r = 0.12 min⁻¹. The fraction of substrate ligated to the ribozyme when T4 RNA ligase was present is also shown (□). Reactions with or without RNA ligase were carried out side by side with the same ribozyme and substrate solutions. (Inset) Electrophoretic analysis of a cleavage reaction carried out with 5′-end labeled substrate in the presence of T4 RNA ligase. Lanes from left to right correspond to the data points in the graph. The migration positions of the substrate-ribozyme covalent complex, uncleaved substrate, circularized substrate, and 5′ cleavage product are shown from top to bottom. Substrate strands are depicted with a thicker line. The residual amount of circularized substrate is the result of an intramolecular ligation reaction catalyzed by T4 RNA ligase on free substrate molecules (23). A similar electrophoretic analysis was carried out in the absence of RNA ligase (not shown).