eIF2ß zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding.

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2ß subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2ß-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2ßS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2ßS264Y mutation has eIF2ß-γ interaction defect. Consistently, the eIF2ßT238A intragenic suppressor mutation restored the eIF2ß-γ and Met-tRNAiMet binding. The eIF2ß-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2ßT238A mutation, suggesting the eIF2ß-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2ßF217A/Q221A double mutation in the HTH domain. The eIF2ßT238A suppressor mutation could not rescue the eIF2ß binding defect of the eIF2γV281K mutation; however, combining the eIF2ßS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2ß with the eIF2γ subunit via its α1-helix, the eIF2ß-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2ß-γ interacts via two distinct binding sites.

Clinical and genetic analysis of essential hypertension with mitochondrial tRNAMet 4435A>G and YARS2 mutation. / 携带线粒体tRNAMet 4435A>Gå’Œæ ¸åŸºå› YARS2çªå˜çš„åŽŸå‘æ€§é«˜è¡€åŽ‹å®¶ç³»é—ä¼ å­¦åˆ†æž.

OBJECTIVES: To investigate the role of m.4435A>G and YARS2 c.572G>T (p.G191V) mutations in the development of essential hypertension. METHODS: A hypertensive patient with m.4435A>G and YARS2 p.G191V mutations was identified from previously collected mitochondrial genome and exon sequencing data. Clinical data were collected, and a molecular genetic study was conducted in the proband and his family members. Peripheral venous blood was collected, and immortalized lymphocyte lines constructed. The mitochondrial transfer RNA (tRNA), mitochondrial protein, adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) in the constructed lymphocyte cell lines were measured. RESULTS: Mitochondrial genome sequencing showed that all maternal members carried a highly conserved m.4435A>G mutation. The m.4435A>G mutation might affect the secondary structure and folding free energy of mitochondrial tRNA and change its stability, which may influence the anticodon ring structure. Compared with the control group, the cell lines carrying m.4435A>G and YARS2 p.G191V mutations had decreased mitochondrial tRNA homeostasis, mitochondrial protein expression, ATP production and MMP levels, as well as increased ROS levels (all P<0.05). CONCLUSIONS: The YARS2 p.G191V mutation aggravates the changes in mitochondrial translation and mitochondrial function caused by m.4435A>G through affecting the steady-state level of mitochondrial tRNA and further leads to cell dysfunction, indicating that YARS2 p.G191V and m.4435A>G mutations have a synergistic effect in this family and jointly participate in the occurrence and development of essential hypertension.

Highly chromophoric fluorescent-labeled methionyl-initiator tRNAs applicable in living cells.

Fluorescent initiator tRNAs (tRNAi) play a crucial role in studying protein synthesis, yet generating highly fluorescent tRNAi complexes remains challenging. We present an optimized strategy to effectively generate highly fluorescent initiator-tRNA complexes in living cells. Our strategy allows the generation of Fluo-Met-tRNAiMet complexes. These complexes can have highly chromogenic N-terminal labeling. For generating such complexes, we use either purified fluorescent methionine (PFM) or non-purified fluorescently labeled methionine (NPFM). Furthermore, PFM promotes the active generation of endogenous tRNAi in cells, leading to highly efficient Fluo-Met-tRNAiMet complexes. Finally, PFM-tRNAiMet complexes also facilitate the visualization of native fluorescently labeled Tat binding to beads. This demonstrates the potential of our approach to advance precision protein engineering and biotechnology applications.

Lamotrigine compromises the fidelity of initiator tRNA recruitment to the ribosomal P-site by IF2 and the RbfA release from 30S ribosomes in Escherichia coli.

Lamotrigine (Ltg), an anticonvulsant drug, targets initiation factor 2 (IF2), compromises ribosome biogenesis and causes toxicity to Escherichia coli. However, our understanding of Ltg toxicity in E. coli remains unclear. While our in vitro assays reveal no effects of Ltg on the ribosome-dependent GTPase activity of IF2 or its role in initiation as measured by dipeptide formation in a fast kinetics assay, the in vivo experiments show that Ltg causes accumulation of the 17S precursor of 16S rRNA and leads to a decrease in polysome levels in E. coli. IF2 overexpression in E. coli increases Ltg toxicity. However, the overexpression of initiator tRNA (i-tRNA) protects it from the Ltg toxicity. The depletion of i-tRNA or overexpression of its 3GC mutant (lacking the characteristic 3GC base pairs in anticodon stem) enhances Ltg toxicity, and this enhancement in toxicity is synthetic with IF2 overexpression. The Ltg treatment itself causes a detectable increase in IF2 levels in E. coli and allows initiation with an elongator tRNA, suggesting compromise in the fidelity/specificity of IF2 function. Also, Ltg causes increased accumulation of ribosome-binding factor A (RbfA) on 30S ribosomal subunit. Based on our genetic and biochemical investigations, we show that Ltg compromises the function of i-tRNA/IF2 complex in ribosome maturation.

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs.

As essential components of the protein synthesis machinery, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of numerous posttranscriptional modifications. Defects in these tRNA maturation steps may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. We previously identified m1A58 as a late modification introduced after modifications Ψ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early during the tRNA modification process, in particular on primary transcripts of initiator tRNAiMet, which prevents its degradation by RNA decay pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined its introduction into yeast elongator and initiator tRNAs. We used specifically modified tRNAs to report on the molecular aspects controlling the Ψ55 → T54 → m1A58 modification circuit in elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that m1A58 has major effects on the structural properties of initiator tRNAiMet, so that the tRNA elbow structure is only properly assembled when this modification is present. This observation provides a structural explanation for the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways.

Translation initiation with exotic amino acids using EF-P-responsive artificial initiator tRNA.

Translation initiation using noncanonical initiator substrates with poor peptidyl donor activities, such as N-acetyl-l-proline (AcPro), induces the N-terminal drop-off-reinitiation event. Thereby, the initiator tRNA drops-off from the ribosome and the translation reinitiates from the second amino acid to yield a truncated peptide lacking the N-terminal initiator substrate. In order to suppress this event for the synthesis of full-length peptides, here we have devised a chimeric initiator tRNA, referred to as tRNAiniP, whose D-arm comprises a recognition motif for EF-P, an elongation factor that accelerates peptide bond formation. We have shown that the use of tRNAiniP and EF-P enhances the incorporation of not only AcPro but also d-amino, ß-amino and γ-amino acids at the N-terminus. By optimizing the translation conditions, e.g. concentrations of translation factors, codon sequence and Shine-Dalgarno sequence, we could achieve complete suppression of the N-terminal drop-off-reinitiation for the exotic amino acids and enhance the expression level of full-length peptide up to 1000-fold compared with the use of the ordinary translation conditions.

NSUN3-mediated mitochondrial tRNA 5-formylcytidine modification is essential for embryonic development and respiratory complexes in mice.

In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.

Ultradeep characterisation of translational sequence determinants refutes rare-codon hypothesis and unveils quadruplet base pairing of initiator tRNA and transcript.

Translation is a key determinant of gene expression and an important biotechnological engineering target. In bacteria, 5'-untranslated region (5'-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. However, the complex interaction of 5'-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. Herein, we systematically assess the dynamic translation from over 1.2 million 5'-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. This allows us to disentangle and precisely quantify effects of various sequence determinants of translation. We find that 5'-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. Moreover, the obtained large-scale data provide clear experimental evidence for a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation.

The initiation factor 3 (IF3) residues interacting with initiator tRNA elbow modulate the fidelity of translation initiation and growth fitness in Escherichia coli.

Initiation factor 3 (IF3) regulates the fidelity of bacterial translation initiation by debarring the use of non-canonical start codons or non-initiator tRNAs and prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain Escherichia coli growth. However, the roles of the N-terminal domain (NTD) have remained unclear. We hypothesized that the interaction between NTD and initiator tRNAfMet (i-tRNA) is essential to coordinate the movement of the two domains during the initiation pathway to ensure fidelity of the process. Here, using atomistic molecular dynamics (MD) simulation, we show that R25A/Q33A/R66A mutations do not impact NTD structure but disrupt its interaction with i-tRNA. These NTD residues modulate the fidelity of translation initiation and are crucial for bacterial growth. Our observations also implicate the role of these interactions in the subunit dissociation activity of CTD of IF3. Overall, the study shows that the interactions between NTD of IF3 and i-tRNA are crucial for coupling the movements of NTD and CTD of IF3 during the initiation pathway and in imparting growth fitness to E. coli.

Initiator tRNA lacking 1-methyladenosine is targeted by the rapid tRNA decay pathway in evolutionarily distant yeast species.

All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G46 (m7G46), N2,N2-dimethylguanosine (m2,2G26), or 4-acetylcytidine (ac4C12), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5'-3' exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m7G46. In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m1A58) specifically target pre-tRNAiMet(CAU) to the nuclear surveillance pathway for 3'-5' exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m1A58 and tRNAiMet(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6Δ mutants lacking m1A58 are temperature sensitive due to decay of tRNAiMet(CAU) by the RTD pathway. Thus, trm6Δ mutants had reduced levels of tRNAiMet(CAU) and not of eight other tested tRNAs, overexpression of tRNAiMet(CAU) restored growth, and spontaneous suppressors that restored tRNAiMet(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNAiMet(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m1A58 in the biology of tRNAiMet(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.

Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

Translation initiation site of mRNA is selected through dynamic interaction with the ribosome.

Initiation of protein synthesis from the correct start codon of messenger RNA (mRNA) is crucial to translation fidelity. In bacteria, the start codon is usually preceded by a 4- to 6-mer adenosine/guanosine-rich Shine­Dalgarno (SD) sequence. Both the SD sequence and the start codon comprise the core ribosome-binding site (RBS), to which the 30S ribosomal subunit binds to initiate translation. How the rather short and degenerate information inside the RBS can be correctly accommodated by the ribosome is not well understood. Here, we used single-molecule techniques to tackle this long-standing issue. We found that the 30S subunit initially binds to mRNA through the SD sequence, whereas the downstream RBS undergoes dynamic motions, especially when it forms structures. The mRNA is either dissociated or stabilized by initiation factors, such as initiation factor 3 (IF3). The initiator transfer RNA (tRNA) further helps the 30S subunit accommodate mRNA and unwind up to 3 base pairs of the RBS structure. Meanwhile, the formed complex of the 30S subunit with structured mRNA is not stable and tends to disassociate. IF3 promotes dissociation by dismissing the bound initiator tRNA. Thus, initiation factors may accelerate the dynamic assembly­disassembly process of 30S­mRNA complexes such that the correct RBS can be preferentially selected. Our study provides insights into how the bacterial ribosome identifies a typical translation initiation site from mRNA.

An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli.

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.

tRNAfMet Inactivating Mycobacterium tuberculosis VapBC Toxin-Antitoxin Systems as Therapeutic Targets.

The Mycobacterium tuberculosis genome contains an abundance of toxin-antitoxin (TA) systems, 50 of which belong to the VapBC family. The activity of VapC toxins is controlled by dynamic association with their cognate antitoxins-the toxin is inactive when complexed with VapB antitoxin but active when freed. Here, we determined the cellular target of two phylogenetically related VapC toxins and demonstrate how their properties can be harnessed for drug development. First, we used a specialized RNA sequencing (RNA-seq) approach, 5' RNA-seq, to accurately identify the in vivo RNA target of M. tuberculosis VapC2 and VapC21 toxins. Both toxins exclusively disable initiator tRNAfMet through cleavage at a single, identical site within their anticodon loop. Consistent with the essential role and global requirement for initiator tRNAfMet in bacteria, expression of each VapC toxin resulted in potent translation inhibition followed by growth arrest and cell death. Guided by previous structural studies, we then mutated two conserved amino acids in the antitoxin (WR→AA) that resided in the toxin-antitoxin interface and were predicted to inhibit toxin activity. Both mutants were markedly less efficient in rescuing growth over time, suggesting that screens for high-affinity small-molecule inhibitors against this or other crucial VapB-VapC interaction sites could drive constitutive inactivation of tRNAfMet by these VapC toxins. Collectively, the properties of the VapBC2 and VapBC21 TA systems provide a framework for development of bactericidal antitubercular agents with high specificity for M. tuberculosis cells.

Systematic evolution of initiation factor 3 and the ribosomal protein uS12 optimizes Escherichia coli growth with an unconventional initiator tRNA.

The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNAcg/GC/cg ) having cg/GC/cg sequence instead of the conventional GC/GC/GC. Both suppressors have the common mutation of V93A in initiation factor 3 (IF3), and additional mutations of either V32L (Sup-1) or H76L (Sup-2) in small subunit ribosomal protein 12 (uS12). The V93A mutation in IF3 was necessary for relaxed fidelity of i-tRNA selection to sustain on i-tRNAcg/GC/cg though with a retarded growth. Subsequent mutations in uS12 salvaged the retarded growth by enhancing the fidelity of translation. The H76L mutation in uS12 showed better fidelity of i-tRNA selection. However, the V32L mutation compensated for the deficient fidelity of i-tRNA selection by ensuring an efficient fidelity check by ribosome recycling factor (RRF). We reveal unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, elongation factor-G (EF-G), RRF, and Pth (peptidyl-tRNA hydrolase) which, taken together, govern the fidelity of translation in bacteria.

Synthesis and properties of the anticodon stem-loop of human mitochondrial tRNAMet containing the disease-related G or m1G nucleosides at position 37.

A single point mutation (A4435G) in the human mitochondrial tRNAMet (hmt-tRNAMet) gene causes severe mitochondrial disorders associated with hypertension, type 2 diabetes and LHON. This mutation leads to the exchange of A37 in the anticodon loop of hmt-tRNAMet for G37 and 1-methylguanosine (m1G37). Here we present the first synthesis and structural/biophysical studies of the anticodon stem and loop of pathogenic hmt-tRNAsMet.

Clinical heterogeneity in patients with m.4412G > A MT-TM mutation and different heteroplasmy levels.

The identification of the m.4412G > A MT-TM (mt-tRNAMet) mutation was first reported in 2019. The affected individual presented with childhood-onset seizures and myopathy and bilateral basal ganglia changes, with heteroplasmy levels in muscle as high as 90%. Here, we describe another adult-onset patient with the same mutation and additional phenotypes, including hearing impairment, cerebellar ataxia, progressive dementia, and myopathy. The 10% heteroplasmy level observed in skin fibroblasts from this patient are lower than those in the previously reported patient. Our report suggests possible clinical heterogeneity in patients with mitochondrial tRNA mutations based on heteroplasmy levels.

Direct Measurements of Erythromycin's Effect on Protein Synthesis Kinetics in Living Bacterial Cells.

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.

Global phosphoproteomics pinpoints uncharted Gcn2-mediated mechanisms of translational control.

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.