tRNA Binding Research Articles

Circadian clock control of interactions between eIF2α kinase CPC-3 and GCN1 with ribosomes regulates rhythmic translation initiation

Misregulation of the activity of GCN2, the kinase that phosphorylates and inactivates translation initiation factor eIF2α, has been implicated in several health disorders, underscoring the need to determine the mechanisms controlling GCN2 activation. During nutrient starvation, increased uncharged tRNA levels trigger GCN1 and GCN20 proteins to mediate the binding of uncharged tRNA to GCN2 to activate the kinase to phosphorylate eIF2α. Under constant conditions, activation of the Neurospora crassa homolog of GCN2, CPC-3, is controlled by the circadian clock. However, how the circadian clock controls the rhythmic activity of CPC-3 was not known. We found that the clock regulates CPC-3 and GCN1 interaction with ribosomes and show that these interactions are necessary for clock regulation of CPC-3 activity. CPC-3 activity rhythms, and the rhythmic interaction of CPC-3 and GCN1 with ribosomes, are abolished in a temperature-sensitive valyl-tRNA synthetase mutant (un-3ts) that has high levels of uncharged tRNAVal at all times of the day. Disrupting the interaction between GCN1 and uncharged tRNA in the absence of GCN20 altered rhythmic CPC-3 activity, indicating that the clock controls the interaction between uncharged tRNA and GCN1. Together, these data support that circadian rhythms in mRNA translation through CPC-3 activity require rhythms in uncharged tRNA levels that drive the rhythmic interaction between CPC-3 and GCN1 with ribosomes. This regulation uncovers a fundamental mechanism to ensure temporal coordination between peak cellular energy levels and the energetically demanding process of mRNA translation.

DDX1 is required for non-spliceosomal splicing of tRNAs but not of XBP1 mRNA

RNA helicase DEAD-box helicase 1 (DDX1) forms a complex with the RNA ligase 2´,3´-cyclic phosphate and 5´-OH ligase (RTCB), which plays a vital role in non-spliceosomal splicing of tRNA and X-box binding protein 1 (XBP1) mRNA. However, the importance of DDX1 in non-spliceosomal splicing has not been clarified. To analyze the functions of DDX1 in mammalian cells, we generated DDX1 cKO cells from the polyploid human U2OS cell line and found that splicing of intron-containing tRNAs was significantly disturbed in DDX1-deficient cells, whereas endoplasmic reticulum (ER) stress-induced splicing of XBP1 mRNA was unaffected. Additionally, the enforced expression of DDX1, but not of its helicase-inactive mutant, rescued the splicing defects of tRNAs in DDX1-deficient cells. These results indicate that RTCB is required for the splicing of both tRNA and XBP1 mRNA, whereas the DDX1 enzymatic activity is specifically required for tRNA splicing in vivo.

TRAMP assembly alters the conformation and RNA binding of Mtr4 and Trf4-Air2

The TRAMP complex contains two enzymatic activities essential for RNA processing upstream of the nuclear exosome. Within TRAMP, RNA is 3' polyadenylated by a subcomplex of Trf4/5 and Air1/2 and unwound 3' to 5' by Mtr4, a DExH helicase. The molecular mechanisms of TRAMP assembly and RNA shuffling between the two TRAMP catalytic sites are poorly understood. Here, we report solution hydrogen-deuterium exchange data with thermodynamic and functional assays to uncover these mechanisms for yeast TRAMP with Trf4 and Air2 homologs. We show that TRAMP assembly constrains RNA-recognition motifs that are peripheral to catalytic sites. These include the Mtr4 Arch and Air2 zinc knuckles 1, 2, and 3. While the Air2 Arch-interacting motif likely constrains the Mtr4 Arch via transient interactions, these do not fully account for the importance of the Mtr4 Arch in TRAMP assembly. We further show that tRNA binding by single active-site subunits, Mtr4 and Trf4-Air2, differs from the double active-site TRAMP. TRAMP has reduced tRNA binding on the Mtr4 Fist and RecA2 domains, offset by increased tRNA binding on Air2 zinc knuckles 2 and 3. Competition between these RNA-binding sites may drive tRNA transfer between TRAMP subunits. We identify dynamic changes upon TRAMP assembly and RNA-recognition motifs that transfer RNA between TRAMP catalytic sites.

Phosphorylation-mediated conformational change regulates human SLFN11

Human Schlafen 11 (SLFN11) is sensitizing cells to DNA damaging agents by irreversibly blocking stalled replication forks, making it a potential predictive biomarker in chemotherapy. Furthermore, SLFN11 acts as a pattern recognition receptor for single-stranded DNA (ssDNA) and functions as an antiviral restriction factor, targeting translation in a codon-usage-dependent manner through its endoribonuclease activity. However, the regulation of the various SLFN11 functions and enzymatic activities remains enigmatic. Here, we present cryo-electron microscopy (cryo-EM) structures of SLFN11 bound to tRNA-Leu and tRNA-Met that give insights into tRNA binding and cleavage, as well as its regulation by phosphorylation at S219 and T230. SLFN11 phosphomimetic mutant S753D adopts a monomeric conformation, shows ATP binding, but loses its ability to bind ssDNA and shows reduced ribonuclease activity. Thus, the phosphorylation site S753 serves as a conformational switch, regulating SLFN11 dimerization, as well as ATP and ssDNA binding, while S219 and T230 regulate tRNA recognition and nuclease activity.

Structure–function analysis of tRNA t6A-catalysis, assembly, and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

The universal N6-threonylcarbamoyladenosine (t6A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t6A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t6A biosynthesis using recombinant TsaC, TsaD–TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD2B2 tetramer. Our TsaD2B2–TsaE–tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1–α2 of TsaD. A. aeolicus TsaD2B2 or TsaDB possesses a basal divalent ion-dependent t6A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t6A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t6A-catalytic activity of TsaD2B2–tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

Determining the Identity of Nucleotides and the Energy of Binding of tRNAs to Their Aminoacyl-tRNA Synthetases Using a Simple Logistic Model

This study showed that the predictor in logistic regression can be applied to estimating the Gibbs free energy of tRNAs’ recognition of and binding to their aminoacyl-tRNA synthetases. Then, 24 linear logistic regression models predicting different classes of tRNAs loaded with a corresponding amino acid were trained in a machine learning classification method, reducing the misclassification error to zero. The models were based on minimal subsets of Boolean explanatory variables describing the favorite presence of nucleotides or nucleosides localized in the different parts of the tRNA. In 90% of cases, they agree with the components of the consensus strand in a class of tRNAs loaded by a given amino acid. According to the proposed theoretical model, the values of the free energy for the entry of the recognition state in the process of tRNA charging were obtained, and the inputs from identity nucleotides and the tRNA strand backbone were distinguished. Almost all the resulting models indicated leading anticodon tandems defining the first and second positions of the anticodon (positions 35 and 36 of the tRNA strand) and the small sets (up to six positions) of the other nucleotides as the natural identity nucleotides most influential in the free energy balance. The magnitude of their input to this energy depends on the position in the strand, favoring positions −1, 35, and 36. The role of position 34 is relatively smaller. These identity attributes may not always be fully arranged in a real single adaptor molecule but were comprehensively present in a given tRNA class. A detailed analysis of the resulting models showed that the absolute value of the energy of binding the tandem 35–36 decreases with the number of identity positions, as well as with the decreasing number of possible hydrogen bonds. On the other hand, in these conditions, the absolute value of the energy of binding of other identity nucleotides increases. All the models indicate that the nucleotide-independent energy of the repulsion tRNA backbone decreases with the number of identity nucleotides. It was also shown that the total free energy change in entering the recognition state increases with the amino acid mass, making this process less spontaneous, which may have an evolutionary reference.

Ribosomes hibernate on mitochondria during cellular stress.

Cell survival under nutrient-deprived conditions relies on cells' ability to adapt their organelles and rewire their metabolic pathways. In yeast, glucose depletion induces a stress response mediated by mitochondrial fragmentation and sequestration of cytosolic ribosomes on mitochondria. This cellular adaptation promotes survival under harsh environmental conditions; however, the underlying mechanism of this response remains unknown. Here, we demonstrate that upon glucose depletion protein synthesis is halted. Cryo-electron microscopy structure of the ribosomes show that they are devoid of both tRNA and mRNA, and a subset of the particles depicted a conformational change in rRNA H69 that could prevent tRNA binding. Our in situ structural analyses reveal that the hibernating ribosomes tether to fragmented mitochondria and establish eukaryotic-specific, higher-order storage structures by assembling into oligomeric arrays on the mitochondrial surface. Notably, we show that hibernating ribosomes exclusively bind to the outer mitochondrial membrane via the small ribosomal subunit during cellular stress. We identify the ribosomal protein Cpc2/RACK1 as the molecule mediating ribosomal tethering to mitochondria. This study unveils the molecular mechanism connecting mitochondrial stress with the shutdown of protein synthesis and broadens our understanding of cellular responses to nutrient scarcity and cell quiescence.

SecM leader peptide as an allosteric translation inhibitor: a molecular dynamics study

The SecM leader peptide regulates translation of the SecA protein, being a part of the Sec translocase, that reversibly arrests the ribosome. In the present study the structure of the SecM complex with the E. coli A/A,P/P–ribosome was obtained by means of docking and molecular dynamics simulation methods. It has been established that binding of the SecM leader peptide in the nascent peptide exit tunnel leads to a turn of the aminoacylating proline residue away from the C–terminal SecM glycine residue, which is adverse to the peptidyltransferase reaction. Besides, the SecM binding leads to a disturbance of the A–tRNA contacts with the tip of the H38 helix of the 23S rRNA (the A–site finger, ASF) and ribosomal protein uL16. Allosteric interrelation between these events has been proved by a construction of networks of concerted changes in non–covalent interactions throughout the whole ribosome, whereupon the A1614 and A751 residues of the 23S rRNA in the exit tunnel that formed stacking interactions with the SecM residues during the MD simulations, were found to be the principal triggers, inducing crucial alterations in the A–tRNA binding. The allosteric signal from the SecM peptide to the ASF, according to our model, is transmitted through ribosomal protein uL22, and there is reason to believe that this sensor is used not only by the SecM leader peptide, but also by other peptides that cause translation arrest.

From sequence to significance: A thorough investigation of the distinctive genome features uncovered in C. Werkmanii strain NIB003

Citrobacter werkmanii (C. werkmanii), an opportunistic urinary bacterium that causes diarrhea, is poorly understood. Our research focuses on genetic features that are crucial to disease development, such as pathogenic interactions, antibiotic resistance, virulence genes and genetic variation. Following its morphological, biochemical, and molecular identification, the whole genome of C. werkmanii strain NIB003 was sequenced in Bangladesh for the first time. Despite having around 80% whole genome conservation, the research shows that the Bangladeshi strain forms a separate phylogenetic cluster. This emphasises the genetic variability within C. werkmanii, resulting in particular modifications at the strain level and changes in its ability to cause disease. The results of the genetic diversity analysis indicate that the Bangladeshi sequenced genome is more diverse than the other strains due to the existence of unique features, such as the presence of t-RNA binding domain and N-6 adenine-specific DNA methylases.

Mechanism of activation of contact-dependent growth inhibition tRNase toxin by the amino acid biogenesis factor CysK in the bacterial competition system.

Contact-dependent growth inhibition (CDI) is a bacterial competition mechanism, wherein the C-terminal toxin domain of CdiA protein (CdiA-CT) is transferred from one bacterium to another, impeding the growth of the toxin recipient. In uropathogenic Escherichia coli 536, CdiA-CT (CdiA-CTEC536) is a tRNA anticodon endonuclease that requires a cysteine biogenesis factor, CysK, for its activity. However, the mechanism underlying tRNA recognition and cleavage by CdiA-CTEC536 remains unresolved. Here, we present the cryo-EM structure of the CysK:CdiA-CTEC536:tRNA ternary complex. The interaction between CdiA-CTEC536 and CysK stabilizes the CdiA-CTEC536 structure and facilitates tRNA binding and the formation of the CdiA-CTEC536 catalytic core structure. The bottom-half of the tRNA interacts exclusively with CdiA-CTEC536 and the α-helices of CdiA-CTEC536 engage with the minor and major grooves of the bottom-half of tRNA, positioning the tRNA anticodon loop at the CdiA-CTEC536 catalytic site for tRNA cleavage. Thus, CysK serves as a platform facilitating the recognition and cleavage of substrate tRNAs by CdiA-CTEC536.

RudS: bacterial desulfidase responsible for tRNA 4-thiouridine de-modification.

In this study, we present an extensiveanalysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicatingthe enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.

Oligomerization and a distinct tRNA-binding loop are important regulators of human arginyl-transferase function

The arginyl-transferase ATE1 is a tRNA-dependent enzyme that covalently attaches an arginine molecule to a protein substrate. Conserved from yeast to humans, ATE1 deficiency in mice correlates with defects in cardiovascular development and angiogenesis and results in embryonic lethality, while conditional knockouts exhibit reproductive, developmental, and neurological deficiencies. Despite the recent revelation of the tRNA binding mechanism and the catalytic cycle of yeast ATE1, the structure-function relationship of ATE1 in higher organisms is not well understood. In this study, we present the three-dimensional structure of human ATE1 in an apo-state and in complex with its tRNA cofactor and a peptide substrate. In contrast to its yeast counterpart, human ATE1 forms a symmetric homodimer, which dissociates upon binding of a substrate. Furthermore, human ATE1 includes a unique and extended loop that wraps around tRNAArg, creating extensive contacts with the T-arm of the tRNA cofactor. Substituting key residues identified in the substrate binding site of ATE1 abolishes enzymatic activity and results in the accumulation of ATE1 substrates in cells.

TRAMP assembly alters the conformation and RNA binding of Mtr4 and Trf4-Air2.

The TRAMP complex contains two enzymatic activities essential for RNA processing upstream of the nuclear exosome. Within TRAMP, RNA is 3' polyadenylated by a sub-complex of Trf4/5 and Air1/2 and unwound 3' to 5' by Mtr4, a DExH helicase. The molecular mechanisms of TRAMP assembly and RNA shuffling between the two TRAMP catalytic sites are poorly understood. Here, we report solution hydrogen-deuterium exchange data with thermodynamic and functional assays to uncover these mechanisms for yeast TRAMP with Trf4 and Air2 homologs. We show that TRAMP assembly constrains RNA-recognition motifs that are peripheral to catalytic sites. These include the Mtr4 Arch and Air2 zinc knuckles 1, 2, and 3. While the Air2 Arch-interacting motif likely constrains the Mtr4 Arch via transient interactions, these do not fully account for the importance of the Mtr4 Arch in TRAMP assembly. We further show that tRNA binding by single active-site subunits, Mtr4 and Trf4-Air2, differs from the double active-site TRAMP. TRAMP has reduced tRNA binding on the Mtr4 Fist and RecA2 domains, offset by increased tRNA binding on Air2 zinc knuckles 2 and 3. Competition between these RNA-binding sites may drive tRNA transfer between TRAMP subunits. We identify dynamic changes upon TRAMP assembly and RNA-recognition motifs that transfer RNA between TRAMP catalytic sites.

Identification of Amino Acids in Trm734 Required for 2'-O-Methylation of the tRNAPhe Wobble Residue.

All organisms methylate their nucleic acids, and this methylation is critical for proper gene expression at both the transcriptional and translational levels. For proper translation in eukaryotes, 2'-O-methylation of C32 (Cm32) and G34 (Gm34) in the anticodon loop of tRNAPhe is critical, with defects in these modifications associated with human disease. In yeast, Cm32 is formed by an enzyme that consists of the methyltransferase Trm7 in complex with the auxiliary protein Trm732, and Gm34 is formed by an enzyme that consists of Trm7 in complex with Trm734. The role of Trm732 and Trm734 in tRNA modification is not fully understood, although previous studies have suggested that Trm734 is important for tRNA binding. In this report, we generated Trm734 variants and tested their ability to work with Trm7 to modify tRNAPhe. Using this approach, we identified several regions of amino acids that are important for Trm734 activity and/or stability. Based on the previously determined Trm7-Trm734 crystal structure, these crucial amino acids are near the active site of Trm7 and are not directly involved in Trm7-Trm734 protein-protein interactions. Immunoprecipitation experiments with these Trm734 variants and Trm7 confirm that these residues are not involved in Trm7-Trm734 binding. Further experiments should help determine if these residues are important for tRNA binding or have another role in the modification of the tRNA. Furthermore, our discovery of a nonfunctional, stable Trm734 variant will be useful in determining if the reported roles of Trm734 in other biological processes such as retromer processing and resistance to Ty1 transposition are due to tRNA modification defects or to other bona fide cellular roles of Trm734.

Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.

Cryo-EM structures of the human Elongator complex at work

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.

High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.