Figure 6. Recombinant CTDK-I complex is active and binds RNA in vitro.
(A) The three-subunit CTDK-I complex from S. cerevisiae was recombinantly expressed in insect cells and purified to homogeneity. The purified complex was run on a 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. (B) Purified human GST-CTD (10 µM) was incubated with 0.4 µM CTDK-I and 3 mM ATP. Time points were taken at 0 (no ATP), 5, 10, 20, 30, 60 and 120 min and CTDK-I activity was determined by western blot analysis using an antibody that recognizes the Ser2 phosphorylated form of the CTD of Pol II. Molecular mass of GST-CTD is ~70 kDa. (C) Purified and dephosporylated Pol II (2 µM) from S. cerevisiae was incubated with 0.4 µM CTDK-I and 3 mM ATP. Time points were taken at 0 (no ATP), 2, 4, 6, 10, 20, 30, 60 and 90 min and CTDK-I activity was determined as in (B). Molecular mass of the CTD containing subunit of Pol II, Rpb1, is ~200 kDa. (D) Increasing concentrations (0–5.8 µM) of the complete CTDK-I kinase complex were incubated with 8 nM of a 24% GC (green line; Kd,app(nM) = 210 ± 18) and with a 45% GC (purple line; Kd,app(nM) = 277 ± 21) ssRNA sequences. Binding was determined by relative change in fluorescence anisotropy. Data was fit with a single site binding equation. Error bars reflect the standard deviation from three experimental replicates. (E) Increasing concentrations (0–5.8 µM) of the complete CTDK-I kinase complex were incubated with 8 nM of a U-rich ssRNA (orange line; Kd,app(nM) = 123 ± 10), an A-rich ssRNA (purple line; Kd,app(nM) = 277 ± 21) and a dsDNA (grey line; Kd,app(nM) = 1007 ± 67) sequences. Binding strength, data fitting and standard deviation was determined as in (D).
Figure 6—figure supplement 1. Recombinant and active CTDK-I complex binds preferentially U-rich ssRNA in vitro.
