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DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5' end or subsequent scanning of the 5' UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn- substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd- substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd- substitutions enhance YKD-KD interactions in vitro, whereas Gcn- substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd- substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.