Figure 4.
Mutational analysis. (A, B) Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2 mM DTT, 10 mM MgCl2, 20 nM 32P-labeled 10-mer HORNA3′p, 100 nM KIN (wild-type or mutant as indicated), and GTP as specified were incubated at 22°C for 10 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. (C) The indicated CalTRL1 alleles on CEN plasmids were tested for S. cerevisiae trl1Δ complementation as described under Methods. +++ signifies growth as well as the wild-type CalTRL1 control at 20, 25, 30 and 37°C.