Effect of YKD substitutions on kinase activity and tRNA binding by purified Gcn2 <i>in vitro</i>.

<p>(<b>A</b>) The indicated Gcn2 proteins (0.25 µg) were incubated with 3 µCi of [γ-³²P]ATP (6000 Ci/mmol, Amersham), 1 µg of recombinant eIF2α−ΔC purified from <i>E. coli</i>, and 0.5 µg of bovine serum albumin in 20 µL of kinase assay buffer (20 mM Tris–HCl [pH 7.9], 50 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol, and 100 µM PMSF) for 5 to 15 min at 30°C. The samples were resolved by 8%–16% SDS–PAGE and subjected to autoradiography. Positions of autophosphorylated Gcn2 (Gcn2-P) and phosphorylated eIF2α−ΔC (eIF2α-P) are indicated. All results were cropped from the same autoradiogram except for those obtained for the R1325E variant, which was analyzed separately with the same WT protein. The results for WT in the latter autoradiogram were essentially identical to those shown here. (<b>B</b>) The extent of Gcn2 autophosphorylation and eIF2α phosphorylation at each time point in (A) was determined by quantifying the intensity of the relevant bands by phosphorimaging of the respective gel bands. Data obtained from 3 independent experiments was averaged and plotted with S.E.M.s as error bars. (<b>C</b>) Purified Gcn2 proteins were incubated at the indicated concentrations with [³²P]-labeled total yeast tRNA in 20 µL of GMSA buffer. Gcn2-tRNA complexes were resolved by electrophoresis through a 1% agarose gel in 1×MOPS buffer (1.5 h, 100 V), transferred to a nitrocellulose membrane and visualized by autoradiography. Unbound [³²P]-tRNA, which has a higher mobility, was present at essentially identical amounts in each lane at levels ∼15-fold higher than the WT Gcn2/tRNA complexes formed at 4 µM (data not shown). All results shown originate from the same gel except for those obtained for the R1325E variant, which was analyzed separately with the same WT protein. The results for WT in the latter gel were similar to those shown here.