Figure 2.

Sequence substitutions at position 638 in the substrate strongly impair cleavage by the VS ribozyme. Products of VS ribozyme cleavage in trans on the substrate with the natural G638, and substitution by A, U or C. Cleavage reactions were performed on radioactively 5′-³²P-labelled substrates under single-turnover conditions at 37°C in standard VS buffer, that is, 50 mM Tris (pH 8.0), 10 mM MgCl₂, 25 mM KCl, 2 mM spermidine. The natural substrate was reacted for 5 min and the three variants for 60 min. Substrate and product were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions and visualized by phosphorimaging. Tracks: 1, G638A; 2, G638U; 3, G638C and 4, G638 natural sequence substrate.