FIGURE 2.
Transcription increases the folding kinetics of the Tetrahymena ribozyme. L-21 ribozyme RNA was incubated in T7 buffer (40 mM TrisCl, pH 7.5, 15 mM MgCl2, 5 mM DTT, and 2 mM spermidine), NTPs, and 10 μg/μL heparin at 37°C (refolding), or transcribed from template DNA under the same conditions (sequential folding). (A) At various times, samples were loaded directly on a native 8% polyacrylamide gel. Band I, folding intermediates; band N, native RNA. The gel was run continuously during the assay so that samples loaded last traveled the shortest distance. (B) Fraction native RNA determined from native gels (fN) versus incubation time was fit to a first-order rate equation. Refolding (open squares) kobs = 0.31 ± 0.2 min−1; sequential folding (filled circles) kobs = 0.51 ± 0.03 min−1.