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. 2017 Nov 20;45(22):12945–12953. doi: 10.1093/nar/gkx1159

Figure 1.

Figure 1.

An autonomous kinase domain of CalTrl1 prefers GTP as the phosphate donor. (A) Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2 mM DTT, 10 mM MgCl2, 100 μM GTP, 20 nM 32P-labeled 10-mer HORNA3′p (shown at bottom, with the 32P-label indicated by •), and 0, 2, 10, 20, 50 or 100 nM CalTrl1 KIN domain were incubated at 22°C for 20 min. The reactions were quenched with an equal volume of 90% formamide, 30 mM EDTA. The labeled RNAs were resolved by urea-PAGE. An autoradiogram of the gel is shown. The positions of the 5′-OH RNA substrate and 5′-PO4 RNA product are indicated on the left. (B–D) Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2 mM DTT, 10 mM MgCl2, 20 nM 10-mer HORNA3′p, 100 nM KIN, and either 0.1, 1, 10 or 100 μM ATP, CTP, GTP or UTP (panel B) or 1, 10 or 100 μM purine NTP analogs (panel D; analog structures shown in panel C) were incubated at 22°C for 2 min. The extents of RNA phosphorylation as a function of NTP concentration are plotted in bar graph format. Each datum is the average of three separate experiments ± SEM.