Fig. 2. RNA in cell lysate is necessary and sufficient for activator dissociation.

a Cdh1 dissociation from APC/C was measured after 45 min in the presence of lysis buffer or yeast lysate, either untreated (Lys) or incubated at 95 °C for 10 min. Prior to the experiment, boiled lysate was gel filtered and concentrated five-fold. Dissociation reactions were performed with or without 5 mM ATP as indicated. Uncropped autoradiograph and source data are provided in the Source Data file. b Purification steps in the preparation of the dissociation activity used in panels c–f. Dissociation activity was found in the flow-through (FT) of the second hydroxyapatite column, which was also eluted with 100 mM phosphate. c Flow-through (FT) and eluate fractions of the last hydroxyapatite step were tested in a Cdh1-dissociation reaction. Fractions were buffer-exchanged into reaction buffer containing 2.5 mM MgCl₂. 5 mM ATP was added as indicated. The vast majority of the dissociation activity was found in the flow-through fraction. Uncropped autoradiograph and source data are provided in the Source Data file. d The hydroxyapatite flow-through fraction was incubated with buffer or Proteinase K (Prot K—6 U/ml) at 37 °C for 1 h. Proteinase K was heat inactivated at 85 °C for 10 min and further inhibited with 1 mM PMSF. After Proteinase K treatment, samples were incubated with buffer or Benzonase (2000 U/ml) at 37 °C for 1 h. Samples were supplemented with 5 mM ATP as indicated. Uncropped autoradiograph and source data are provided in the Source Data file. e Nucleic acid species in the flow-through fraction were extracted with phenol–chloroform, separated by a 10% TBE urea polyacrylamide gel and stained with SYBR Safe. RNase A treatment (0.2 mg/ml) was performed at 37 °C for 30 min (right lane). f Phenol–chloroform-extracted RNA species from panel e were tested for Cdh1 dissociation with or without 3 mM ATP in the presence of a reaction buffer containing 2.5 mM MgCl₂. RNase A treatment (0.2 mg/ml) was performed at 37 °C for 30 min (right lane). Uncropped autoradiograph and source data are provided in the Source Data file.