A subset of Gcn^- substitutions of conserved surface-exposed residues in the HisRS domain impair kinase activity but not tRNA binding by purified Gcn2 in vitro.

<p><b>(A, B and C)</b> The indicated purified Gcn2 proteins were incubated with [³²P]-labeled total yeast tRNA in 20 μL of GMSA buffer. The Gcn2-tRNA complexes were resolved by electrophoresis through a 1% agarose gel in 1×MOPS buffer (1.5h, 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 4μM. The purified Gcn2 proteins used in the assays were visualized by staining with Coomassie brilliant blue following separation by SDS-PAGE (images labelled Coomassie). <b>(D)</b> The indicated Gcn2 proteins (ca. 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</i>. <i>coli</i>, and 0.5 μg of bovine serum albumin in 20 μL of kinase assay buffer at 30^°C for the indicated times. 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.