Gcn^- substitutions D1327A/D1327K strengthen HisRS/CTD domain interaction in a manner antagonized by uncharged tRNA.

<p><b>(A)</b> Yeast two-hybrid analysis of domain interactions. Strain EGY48 carrying the <i>lexAop-lacZ</i> reporter plasmid was co-transformed with plasmids encoding lexA-HisRS (Gcn2 residues 970–1497) (with or without the indicated substitutions) and B42-CTD (Gcn2 residues 1497–1659). Strains were cultured and β-galactosidase activities were measured in WCEs. <b>(B)</b><i>In vitro</i> binding of LexA-CTD (Gcn2 residues 1498–1659), expressed in yeast, to GST fusion proteins containing the Gcn2 HisRS domain (residues 970–1497), either WT or containing the indicating mutations. Aliquots of nuclease-treated whole-cell extracts (500 μg of total protein) from transformants of yeast <i>gcn2</i>Δ strain HQY132 carrying plasmids containing the LexA-CTD fusion protein were incubated with approximately equivalent amounts of GST or GST-HisRS (970–1497) fusion proteins immobilized on glutathione-Sepharose beads. After extensive washing, LexA fusion proteins bound to the GST proteins were resolved by SDS-PAGE and detected by immunoblot analysis with anti-LexA (Top) and anti-GST (bottom) antibodies. Results from quantification of LexA blots from replicate experiments are summarized in the histogram below as mean percentages of input amounts recovered in the pull-downs. <b>(C)</b> Yeast two-hybrid analysis as shown in (A). Samples for β-galactosidase assay were collected from cells under starvation (+) or nonstarvation (-) conditions as described in Materials and Methods. The results shown are averages of activities from three or more individual transformants. <b>(D)</b> Yeast two-hybrid analysis of CTD and KD interaction domains in Gcn2. Plasmids pHQ311 and pHQ428 encoding respectively the lexA-CTD(1498–1659) and B42-KD(720–999) segments were co-transformed into strain EGY48 carrying the <i>lexAop-lacZ</i> reporter plasmid. Samples for β-galactosidase assay were collected from cells under starvation (+) or nonstarvation (-) conditions. The results shown are averages of activities from three or more individual transformants. <b>(E and F)</b><i>In vitro</i> binding of Gcn2 [³⁵S]-HisRS(970–1497) segment translated <i>in vitro</i> to GST fusion proteins containing the Gcn2 KD (568–998), CTD (1498–1659) or HisRS (970–1497) domains. RNA (tRNA^Phe or mRNA) was added to the reaction to the indicated final concentration. The mean fold-reductions in pull-downs of [³⁵S]-HisRS(970–1497) on tRNA^Phe addition were calculated from replicate experiments and presented in the histogram below the gel.