Skip to main content
. 2018 May;24(5):721–738. doi: 10.1261/rna.065037.117

FIGURE 8.

FIGURE 8.

Grc3 PNK active site motifs are critical for kinase activity in vitro. (A) SDS-PAGE analysis of recombinant Sc Las1 bound to Grc3 WT, P-loop, Walker B, Clasp, and Lid variants. (B) Melting temperature of Grc3/Las1 PNK variants determined by thermal shift experiments. The mean and standard deviation were calculated from three independent replicates. (C) RNA phosphorylation of 0–2 µM Grc3/Las1 variants incubated with 21-nt ssRNA (15 µM). Specific activity was calculated from the moles of phosphorylated RNA in the 60 min PNK reaction per mole of input Grc3/Las1 in the linear range of the protein titration. (D) SDS-PAGE analysis of recombinant Sc Las1 bound to Grc3 WT, and single P-loop, Walker B, Clasp, and Lid variants. (E) RNA phosphorylation of 16 µM Grc3/Las1 variants incubated with 21-nt ssRNA (10 µM). The mean and standard deviation were calculated from three independent replicates. (*) P < 5 × 10−4; (**) P < 2 × 10−4; n.s., not significant were calculated from two-tailed Student's t-tests.