Novel mitochondrial tRNALeu(UUR) 3261A > g mutation in two pedigrees with essential hypertension.

BACKGROUND: Essential hypertension (EH) was associated with mitochondrial tRNA mutations. AIMS: This study was designed to assess the association between EH and mitochondrial dysfunction. METHODS: A total of 30 individuals from two different Chinese families exhibit maternally inherited EH were assessed for genetic, clinical, and biochemical phenotypes pertaining to EH and mitochondrial functionality. These analyses included assessments of tRNALeu(UUR) 3261A > G mutation status, mitochondrial membrane permeability, mitochondria-associated ATP and reactive oxygen species (ROS) generation, and electron transport chain functionality. RESULTS: EH was detected in 6 total analyzed members of the two families assessed in the present study, with its initial age of onset and presentation varying among patients. These patients with EH exhibited the tRNALeu(UUR) 3261A > G mutation and were of the B5 and D4 Eastern Asian mitochondrial haplogroups. This 3261A > G mutation was predicted to result in disruption of normal tRNALeu(UUR) activity owing to the destabilization of conserved base pairing (30A-40U). Consistent with this prediction, we found that cybrid cell lines exhibiting this 3261A > G mutation exhibited a ~49.05% decrease in baseline tRNALeu(UUR) levels. These cells additionally exhibited ~44.81% reductions in rates of mitochondrial translation. CONCLUSIONS: To facilitate future molecular diagnosis, the 3261A > G mutation should be included in the list of hereditary risk factors. Our findings will aid in the counseling of EH families.

Mitochondrial genome variant m.3250T>C as a possible risk factor for mitochondrial cardiomyopathy.

The MT-TL1 gene codes for the mitochondrial leucine transfer RNA (tRNALeu(UUR) ) necessary for mitochondrial translation. Pathogenic variants in the MT-TL1 gene result in mitochondriopathy in humans. The m.3250T>C variant in the MT-TL1 gene has been previously associated with exercise intolerance and mitochondrial myopathy, yet disease classification for this variant has not been consistently reported. Molecular studies suggest the m.3250T>C variant does not alter tRNALeu(UUR) structure but may have a modest impact on aminoacylation capacity. However, functional studies are limited. Our study aimed to further define the clinical presentation, inheritance pattern, and molecular pathology of the m.3250T>C variant. Families with the m.3250T>C variant were recruited from the Mitochondrial Disease Clinic at Cincinnati Children's Hospital Medical Center and GeneDx laboratory database. Affected individuals most frequently presented with cardiac findings, exercise intolerance, and muscle weakness. Hypertrophic cardiomyopathy was the most frequent cardiac finding. Many asymptomatic individuals had homoplasmic or near homoplasmic levels of the m.3250T>C variant, suggesting the penetrance is incomplete. Patient-derived fibroblasts demonstrated lowered ATP production and increased levels of reactive oxygen species. Our results demonstrate that the m.3250T>C variant exhibits incomplete penetrance and may be a possible cause of cardiomyopathy by impacting cellular respiration in mitochondria.

Fragmented mitochondrial genomes evolved in opposite directions between closely related macaque louse Pedicinus obtusus and colobus louse Pedicinus badii.

We report for the first time the fragmented mitochondrial (mt) genomes of two Pedicinus species: Pedicinus obtusus and Pedicinus badii, and compared them with the lice of humans and chimpanzees. Despite being congeneric, the two monkey lice are distinct from each other in mt karyotype. The variation in mt karyotype between the two Pedicinus lice is the most pronounced among the congeneric species of sucking lice observed to date and is attributable to the opposite directions between them in mt karyotype evolution. Two of the inferred ancestral mt minichromosomes of the higher primate lice merged as one in the macaque louse whereas one of the ancestral minichromosomes split into two in the colobus louse after these two species diverged from their most recent common ancestor. Our results showed that mt genome fragmentation was a two-way process in the higher primate lice, and minichromosome merger was more common than previously thought.

Deciphering the interaction of benzoxaborole inhibitor AN2690 with connective polypeptide 1 (CP1) editing domain of Leishmania donovani leucyl-tRNA synthetase.

Leucyl-tRNA synthetases (LRS) catalyze the linkage of leucine with tRNALeu. A large insertion CP1 domain (Connective Polypeptide 1) in LRS is responsible for post-transfer editing of mis-charged aminoacyl-tRNAs. Here, we characterized the CP1 domain of Leishmania donovani, a protozoan parasite, and its role in editing activity and interaction with broad spectrum anti-fungal, AN2690. The deletion mutant of LRS, devoid of CP1 domain (LRS-CP1Δ) was constructed, followed by determination of its role in editing and aminoacylation. Binding of AN2690 and different amino acids with CP1 deletion mutant and full length LRS was evaluated using isothermal titration calorimetry (ITC) and molecular dynamics simulations. The recombinant LRS-CP1Δ protein did not catalyze the aminoacylation and the editing reaction when compared to full-length LRS. Thus, indicating that CP1 domain was imperative for both aminoacylation and editing activities of LRS. Binding studies with different amino acids indicated selectivity of isoleucine by CP1 domain over other amino acids. These studies also indicated high affinity of AN2690 with the editing domain. Molecular docking studies indicated that AN2690-CP1 domain complex was stabilized by hydrogen bonding and hydrophobic interactions resulting in high binding affinity between the two. Our data suggests CP1 is crucial for the function of L.donovani LRS.

LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor.

Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells.

RNA 3' polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to high levels of the intercalating agent ethidium bromide (EtBr), greater than those required to suppress mitochondrial transcription, mitochondrial tRNAs in human cells became polyadenylated. Relaxation of the inducing stress led to rapid turnover of the polyadenylated tRNAs. The extent, kinetics and duration of tRNA polyadenylation were EtBr dose-dependent, with mitochondrial tRNAs differentially sensitive to the stress. RNA interference and inhibitor studies indicated that ongoing mitochondrial ATP synthesis, plus the mitochondrial poly(A) polymerase and SUV3 helicase were required for tRNA polyadenylation, while polynucleotide phosphorylase counteracted the process and was needed, along with SUV3, for degradation of the polyadenylated tRNAs. Doxycycline treatment inhibited both tRNA polyadenylation and turnover, suggesting a possible involvement of the mitoribosome, although other translational inhibitors had only minor effects. The dysfunctional tRNALeu(UUR) bearing the pathological A3243G mutation was constitutively polyadenylated at a low level, but this was markedly enhanced after doxycycline treatment. We propose that polyadenylation of structurally and functionally abnormal mitochondrial tRNAs entrains their PNPase/SUV3-mediated destruction, and that this pathway could play an important role in mitochondrial diseases associated with tRNA mutations.

A hypertension-associated mitochondrial DNA mutation alters the tertiary interaction and function of tRNALeu(UUR).

Several mitochondrial tRNA mutations have been associated with hypertension, but their pathophysiology remains poorly understood. In this report, we identified a novel homoplasmic 3253T→C mutation in the mitochondrial tRNALeu(UUR) gene in a Han Chinese family with maternally inherited hypertension. The m.3253T→C mutation affected a highly conserved uridine at position 22 at the D-stem of tRNALeu(UUR), introducing a G-C base pairing (G13-C22) at the D-stem and a tertiary base pairing (C22-G46) between the D-stem and the variable loop. We therefore hypothesized that the m.3253T→C mutation altered both the structure and function of tRNALeu(UUR) Using cytoplasmic hybrid (cybrid) cell lines derived from this Chinese family, we demonstrated that the m.3253T→C mutation perturbed the conformation and stability of tRNALeu(UUR), as suggested by faster electrophoretic mobility of mutated tRNA relative to the wild-type molecule. Northern blot analysis revealed an ∼45% decrease in the steady-state level of tRNALeu(UUR) in the mutant cell lines carrying the m.3253T→C mutation, as compared with control cell lines. Moreover, an ∼35% reduction in aminoacylation efficiency of tRNALeu(UUR) was observed in the m.3253T→C mutant cells. These alterations in tRNALeu(UUR) metabolism impaired mitochondrial translation, especially for those polypeptides with a high proportion of Leu(UUR) codons, such as ND6. Furthermore, we demonstrated that the m.3253T→C mutation decreased the activities of mitochondrial complexes I and V, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the cells. In conclusion, our findings may provide new insights into the pathophysiology of maternally inherited hypertension.

Structural and biochemical insights into the 2'-O-methylation of pyrimidines 34 in tRNA.

tRNA molecules undergo extensive modifications during their maturation and these modifications play important cellular roles. TrmL is a tRNA-modification enzyme that catalyzes the transfer of a methyl group to the 2'-hydroxyl group of the pyrimidines at the wobble position 34 in two tRNALeu isoacceptors, but the mechanism remains elusive. In this study, we determined the crystal structure of TrmL from Thermus thermophilus (TtTrmL) to 1.7 Å. The enzyme contains only the conserved minimal SPOUT fold, but displays distinct biochemical behavior from its Escherichia coli counterpart, EcTrmL. Interestingly, a fortuitous ligand of 5'-methylthioadenosine was consistently found at the SAM-binding pocket in the crystal structures, which probably came from the expression host. Both TtTrmL and EcTrmL were capable of methylating each other's tRNA substrates, but the latter exhibited much higher activity than the former. Enzymatic activity assays showed that the reaction catalyzed by TtTrmL greatly depends on the reaction pH and is also affected by salt concentration. Via sequence alignment and structural superposition, we discovered that a universally conserved glutamate residue is likely to fulfill the role of the general base for the initial proton abstraction from the 2'-hydroxyl group of pyrimidines 34. Lastly, based on our structural and biochemical data, we proposed the dimer interface to be the tRNA-binding site for TtTrmL. DATABASE: The atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession number 5CO4.

Idiosyncratic recognition of UUG/UUA codons by modified nucleoside 5-taurinomethyluridine, τm5U present at 'wobble' position in anticodon loop of tRNALeu: A molecular modeling approach.

Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the 'wobble' 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by 'wobble' as well as a novel 'single' hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons.

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu.

Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNA(Leu) formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNA(Leu) through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNA(Ser) (CatRNA(Ser)(CAG)) in addition to its cognate CatRNA(Leu), leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNA(Ser), suggesting the significance of their highly divergent CTDs in tRNA(Ser) recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNA(Ser) and cognate CatRNA(Leu). Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain.

Fragmented mitochondrial genomes in two suborders of parasitic lice of eutherian mammals (Anoplura and Rhynchophthirina, Insecta).

Parasitic lice (order Phthiraptera) infest birds and mammals. The typical animal mitochondrial (mt) genome organization, which consists of a single chromosome with 37 genes, was found in chewing lice in the suborders Amblycera and Ischnocera. The sucking lice (suborder Anoplura) known, however, have fragmented mt genomes with 9-20 minichromosomes. We sequenced the mt genome of the elephant louse, Haematomyzus elephantis - the first species of chewing lice investigated from the suborder Rhynchophthirina. We identified 33 mt genes in the elephant louse, which were on 10 minichromosomes. Each minichromosome is 3.5-4.2 kb in size and has 2-6 genes. Phylogenetic analyses of mt genome sequences confirm that the elephant louse is more closely related to sucking lice than to the chewing lice in the Amblycera and Ischnocera. Our results indicate that mt genome fragmentation is shared by the suborders Anoplura and Rhynchophthirina. Nine of the 10 mt minichromosomes of the elephant louse differ from those of the sucking lice (Anoplura) known in gene content and gene arrangement, indicating that distinct mt karyotypes have evolved in Anoplura and Rhynchophthirina since they diverged ~92 million years ago.

Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2'-O-methyltransferase.

Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNA(Leu)CAA and tRNA(Leu)UAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2'-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i(6)) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2'-O-methylation relies on prior N(6)-isopentenyladenosine modification at position 37.

Zinc is the molecular "switch" that controls the catalytic cycle of bacterial leucyl-tRNA synthetase.

The Escherichia coli (E. coli) leucyl-tRNA synthetase (LeuRS) enzyme is part of the aminoacyl-tRNA synthetase (aaRS) family. LeuRS is an essential enzyme that relies on specialized domains to facilitate the aminoacylation reaction. Herein, we have biochemically characterized a specialized zinc-binding domain 1 (ZN-1). We demonstrate that the ZN-1 domain plays a central role in the catalytic cycle of E. coli LeuRS. The ZN-1 domain, when associated with Zn(2+), assumes a rigid architecture that is stabilized by thiol groups from the residues C159, C176 and C179. When LeuRS is in the aminoacylation complex, these cysteine residues form an equilateral planar triangular configuration with Zn(2+), but when LeuRS transitions to the editing conformation, this geometric configuration breaks down. By generating a homology model of LeuRS while in the editing conformation, we conclude that structural changes within the ZN-1 domain play a central role in LeuRS's catalytic cycle. Additionally, we have biochemically shown that C159, C176 and C179 coordinate Zn(2+) and that this interaction is essential for leucylation to occur, but is not essential for deacylation. Furthermore, calculated Kd values indicate that the wild-type enzyme binds Zn(2+) to a greater extent than any of the mutant LeuRSs. Lastly, we have shown through secondary structural analysis of our LeuRS enzymes that Zn(2+) is an architectural cornerstone of the ZN-1 domain and that without its geometric coordination the domain collapses. We believe that future research on the ZN-1 domain may reveal a possible Zn(2+) dependent translocation mechanism for charged tRNA(Leu).

Synthesis of 3'-triazoyl-dinucleotides as precursors of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues.

We report here the synthesis of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues containing a 1,2,3-triazole ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycloaddition of alkyne Phe and Leu analogues to 3'-azido-3'-deoxyadenosine via the Cu(I)-catalysed Huisgen, Meldal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable Phe-tRNA(Phe) analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of aminoacyl-tRNA/protein complexes.

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase.

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.

Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei.

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNA(Phe) is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix α4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNA(Leu) isoacceptors as a second substrate. We show that the G(1)-C(72) pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop α6-α7 or D205 located in the loop ß6-α8 affected Leu-tRNA(Leu) isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites.

Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.

The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL.

Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.

Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

Interdomain communication modulates the tRNA-dependent pre-transfer editing of leucyl-tRNA synthetase.

EcLeuRS [Escherichia coli LeuRS (leucyl-tRNA synthetase)] has evolved both tRNA-dependent pre- and post-transfer editing capabilities to ensure catalytic specificity. Both editing functions rely on the entry of the tRNA CCA tail into the editing domain of the LeuRS enzyme, which, according to X-ray crystal structural studies, leads to a dynamic disordered orientation of the interface between the synthetic and editing domains. The results of the present study show that this tRNA-triggered conformational rearrangement leads to interdomain communication between the editing and synthetic domains through their interface, and this communication mechanism modulates the activity of tRNA-dependent pre-transfer editing. Furthermore, tRNA-dependent editing reaction inhibits misactivating non-cognate amino acids from the synthetic active site. These results also suggested a novel quality control mechanism of EcLeuRS which is achieved through the co-ordination between the synthetic and editing domains.