CYT-19 dissociation from the ribozyme.

<p>Following a CYT-19 washout in the continued presence of 2 mM AMP–PNP, the average FRET value was followed for ribozyme molecules that started this observation period with the P1 helix undocked (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>). The time evolution of the average FRET value for these molecules (red, 62 molecules) was fit by three phases with rate constants and relative amplitudes of 30 min⁻¹ (0.36), 0.43 min⁻¹ (0.29), and 0.01 min⁻¹ (0.35). We infer that the rate constant of 0.43 min⁻¹ reflects CYT-19 dissociation because this phase was not observed in the absence of CYT-19. The initial fast phase reflects P1 docking re-equilibration with bound CYT-19 and is predicted from the model, and the slowest phase most likely reflects the slow conversion of ribozyme molecules that initially give poor docking or are misfolded (see Results, “CYT-19 Can Remain Associated with the Ribozyme for Multiple Cycles of Helix Capture”). In the absence of CYT-19 (black, 64 molecules), re-equilibration of P1 docking gave a single observed phase of 130 min⁻¹ (inset). The endpoint is lower (0.73) than expected (0.85, indicated by dashed line), most likely reflecting molecules that dock P1 poorly as above.