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

FIGURE 1.

FIGURE 1.

Characterization of recombinant Grc3/Las1 PNK activity. (A) RNA phosphorylation activity of Grc3/Las1 (2 µM) with ssRNA (15 µM) in the presence of 5 mM EDTA or 15 mM divalent metal [MgCl2, MnCl2, Ca(OAc)2, Zn(OAc)2, NiCl2]. A representative gel is shown (above) where P► marks phosphorylated RNA and sample “x” is an RNA alone control. (B) RNA phosphorylation activity of Grc3/Las1 (2 µM) with ssRNA (15 µM) in the presence of an assortment of nucleotides (ATP, UTP, CTP, GTP, dATP, dTTP, dCTP, and dGTP at 1 mM). A representative gel is shown (above) where P► marks phosphorylated RNA and sample “x” is a no nucleotide control. (C) Nucleotide concentration dependence of RNA phosphorylation. Grc3/Las1 (2 µM) was incubated with 15 µM fluorescently labeled ssRNA in the presence of 0–250 µM ATP, dATP, CTP, GTP, and UTP. (D) Data from C was fit using a Michaelis–Menten linear regression to calculate the Michaelis constant (Km) and catalytic constant (Kcat). The mean of three independent replicates were plotted with the standard deviation.