Association between Archaeal prolyl- and leucyl-tRNA synthetases enhances tRNA(Pro) aminoacylation

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA(Pro) charging showed that the catalytic efficiency (k(cat)/K(m)) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K(m) for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.     

Structural and functional mapping of the archaeal multi-aminoacyl-tRNA synthetase complex

Methanothermobacter thermautotrophicus contains a multi-aminoacyl-tRNA synthetase complex (MSC) of LysRS, LeuRS and ProRS. Elongation factor (EF) 1A also associates to the MSC, with LeuRS possibly acting as a core protein. Analysis of the MSC revealed that LysRS and ProRS specifically interact with the idiosyncratic N- and C- termini of LeuRS, respectively. EF-1A instead interacts with the inserted CP1 proofreading domain, consistent with models for post-transfer editing by class I synthetases such as LeuRS. Together with previous genetic data, these findings show that LeuRS plays a central role in mediating interactions within the archaeal MSC by acting as a core scaffolding protein.     

An aminoacyl-tRNA synthetase:elongation factor complex for substrate channeling in archaeal translation

Translation requires the specific attachment of amino acids to tRNAs by aminoacyl-tRNA synthetases (aaRSs) and the subsequent delivery of aminoacyl-tRNAs to the ribosome by elongation factor 1 alpha (EF-1α). Interactions between EF-1α and various aaRSs have been described in eukaryotes, but the role of these complexes remains unclear. To investigate possible interactions between EF-1α and other cellular components, a yeast two-hybrid screen was performed for the archaeon Methanothermobacter thermautotrophicus. EF-1α was found to form a stable complex with leucyl-tRNA synthetase (LeuRS; K_D = 0.7 μM). Complex formation had little effect on EF-1α activity, but increased the k_cat for Leu-tRNA^Leu synthesis ~8-fold. In addition, EF-1α co-purified with the archaeal multi-synthetase complex (MSC) comprised of LeuRS, LysRS and ProRS, suggesting the existence of a larger aaRS:EF-1α complex in archaea. These interactions between EF-1α and the archaeal MSC contribute to translational fidelity both by enhancing the aminoacylation efficiencies of the three aaRSs in the complex and by coupling two stages of translation: aminoacylation of cognate tRNAs and their subsequent channeling to the ribosome.     

Mechanism of the activation step of the aminoacylation reaction: a significant difference between class I and class II synthetases

In the present work we report, for the first time, a novel difference in the molecular mechanism of the activation step of aminoacylation reaction between the class I and class II aminoacyl tRNA synthetases (aaRSs). The observed difference is in the mode of nucleophilic attack by the oxygen atom of the carboxylic group of the substrate amino acid (AA) to the αP atom of adenosine triphosphate (ATP). The syn oxygen atom of the carboxylic group attacks the α-phosphorous atom (αP) of ATP in all class I aaRSs (except TrpRS) investigated, while the anti oxygen atom attacks in the case of class II aaRSs. The class I aaRSs investigated are GluRS, GlnRS, TyrRS, TrpRS, LeuRS, ValRS, IleRS, CysRS, and MetRS and class II aaRSs investigated are HisRS, LysRS, ProRS, AspRS, AsnRS, AlaRS, GlyRS, PheRS, and ThrRS. The variation of the electron density at bond critical points as a function of the conformation of the attacking oxygen atom measured by the dihedral angle ψ (C(α)-C') conclusively proves this. The result shows that the strength of the interaction of syn oxygen and αP is stronger than the interaction with the anti oxygen for class I aaRSs. This indicates that the syn oxygen is the most probable candidate for the nucleophilic attack in class I aaRSs. The result is further supported by the computation of the variation of the nonbonded interaction energies between αP atom and anti oxygen as well as syn oxygen in class I and II aaRSs, respectively. The difference in mechanism is explained based on the analysis of the electrostatic potential of the AA and ATP which shows that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs, which is correlated with the organization of the active site in respective aaRSs. A comparative study of the reaction mechanisms of the activation step in a class I aaRS (Glutaminyl tRNA synthetase) and in a class II aaRS (Histidyl tRNA synthetase) is carried out by the transition state analysis. The atoms in molecule analysis of the interaction between active site residues or ions and substrates are carried out in the reactant state and the transition state. The result shows that the observed novel difference in the mechanism is correlated with the organizations of the active sites of the respective aaRSs. The result has implication in understanding the experimentally observed different modes of tRNA binding in the two classes of aaRSs.     

An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA synthetase (MtArgRS) as an interacting partner of MtSerRS. Surface plasmon resonance confirmed stable complex formation, with a dissociation constant (K(D)) of 250 nM. Formation of the MtSerRS·MtArgRS complex was further supported by the ability of GST-MtArgRS to co-purify MtSerRS and by coelution of the two enzymes during gel filtration chromatography. The MtSerRS·MtArgRS complex also contained tRNA(Arg), consistent with the existence of a stable ribonucleoprotein complex active in aminoacylation. Steady-state kinetic analyses revealed that addition of MtArgRS to MtSerRS led to an almost 4-fold increase in the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that formation of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions.     

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNALys

Aminoacyl-tRNA synthetases(aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs). Previously, two mutations(c.1129 GA and c.517 TC) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNA~(Lys), with the c.517TC mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129GA and c.517TC mutations and provided yeast genetic bases to investigate other KARS mutations in the future.     

An aminoacyl-tRNA synthetase with a defunct editing site

Many aminoacyl-tRNA synthetases (aaRSs) contain two active sites, a synthetic site catalyzing aminoacyl-adenylate formation and tRNA aminoacylation and a second editing or proofreading site that hydrolyzes misactivated adenylates or mischarged tRNAs. The combined activities of these two sites lead to rigorous accuracy in tRNA aminoacylation, and both activities are essential to LeuRS and other aaRSs. Here, we describe studies of the human mitochondrial (hs mt) LeuRS indicating that the two active sites of this enzyme have undergone functional changes that impact how accurate aminoacylation is achieved. The sequence of the hs mt LeuRS closely resembles a bacterial LeuRS overall but displays significant variability in regions of the editing site. Studies comparing Escherichia coli and hs mt LeuRS reveal that the proofreading activity of the mt enzyme is disrupted by these sequence changes, as significant levels of Ile-tRNA(Leu) are formed in the presence of high concentrations of the noncognate amino acid. Experiments monitoring deacylation of Ile-tRNA(Leu) and misactivated adenylate turnover revealed that the editing active site is not operational. However, hs mt LeuRS has weaker binding affinities for both cognate and noncognate amino acids relative to the E. coli enzyme and an elevated discrimination ratio. Therefore, the enzyme achieves fidelity using a more specific synthetic active site that is not prone to errors under physiological conditions. This enhanced specificity must compensate for the presence of a defunct editing site and ensures translational accuracy.     

Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases

Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (approximately 180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus. This non-cognate amino acid was efficiently acylated in vitro onto tRNA(Pro), and the misacylated Cys-tRNA(Pro) was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.     

Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex

Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.     

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.     

Molecular and functional dissection of a putative RNA-binding region in yeast mitochondrial leucyl-tRNA synthetase

Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the beta-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS-RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex.     

Anticodon recognition and discrimination by the alpha-helix cage domain of class I lysyl-tRNA synthetase

Aminoacyl-tRNA synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-tRNA synthetase which exists in both classes I (LysRS1) and II (LysRS2). Differences in tRNA acceptor stem recognition between LysRS1 and LysRS2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of tRNA anticodon recognition by LysRS1. On the basis of structure-based sequence alignments, seven tRNALys anticodon variants and seven LysRS1 anticodon binding site variants were selected for analysis of the Pyrococcus horikoshii LysRS1-tRNALys docking model. LysRS1 specifically recognized the bases at positions 35 and 36, but not that at position 34. Aromatic residues form stacking interactions with U34 and U35, and aminoacylation kinetics also identified direct interactions between Arg502 and both U35 and U36. Tyr491 was also found to interact with U36, and the Y491E variant exhibited significant improvement compared to the wild type in aminoacylation of a tRNALysUUG mutant. Refinement of the LysRS1-tRNALys docking model based upon these data suggested that anticodon recognition by LysRS1 relies on considerably fewer interactions than that by LysRS2, providing a structural basis for the more significant role of the anticodon in tRNA recognition by the class II enzyme. To date, only glutamyl-tRNA synthetase (GluRS) has been found to contain an alpha-helix cage anticodon binding domain homologous to that of LysRS1, and these data now suggest that specificity for the anticodon of tRNALys could have been acquired through relatively few changes to the corresponding domain of an ancestral GluRS enzyme.     

The C-terminal domain of the archaeal leucyl-tRNA synthetase prevents misediting of isoleucyl-tRNA(Ile)

In the archaeal leucyl-tRNA synthetase (LeuRS), the C-terminal domain recognizes the long variable arm of tRNA(Leu) for aminoacylation, and the so-called editing domain deacylates incorrectly formed Ile-tRNA(Leu). We previously reported, for Pyrococcus horikoshii LeuRS, that a deletion mutant lacking the C-terminal domain (LeuRS_delta(811-967)) retains normal editing activity, but has severely reduced aminoacylation activity. In this study, we found that LeuRS_delta(811-967), but not the wild-type LeuRS, exhibited surprisingly robust deacylation activity against Ile-tRNA(Ile), correctly formed by isoleucyl-tRNA synthetase ("misediting"). Structural superposition of tRNA(Ile) onto the LeuRS x tRNA(Leu) complex indicated that Ile911, Lys912, and Glu913 of the LeuRS C-terminal domain clash with U20 of tRNA(Ile), which is bulged out as compared to the corresponding nucleotide of tRNA(Leu). The deletion of amino acid residues 911-913 of LeuRS enhanced the Ile-tRNA(Ile) deacylation activity, without affecting the Ile-tRNA(Leu) deacylation activity. These results demonstrate that the clashing between U20 of tRNA(Ile) and residues 911-913 of the LeuRS C-terminal domain is the structural mechanism that prevents misediting. In contrast, the deletion of the C-terminal domains of the isoleucyl- and valyl-tRNA synthetases impaired both the aminoacylation (Ile-tRNA(Ile) and Val-tRNA(Val) formation, respectively) and editing (Val-tRNA(Ile) and Thr-tRNA(Val) deacylation, respectively) activities, and did not cause misediting (Val-tRNA(Val) and Thr-tRNA(Thr) deacylation, respectively) activity. Thus, the requirement of the C-terminal domain for misediting prevention is unique to LeuRS, which does not recognize the anticodon of the cognate tRNA, unlike the common aminoacyl-tRNA synthetases.     

Functional guanine-arginine interaction between tRNAPro and prolyl-tRNA synthetase that couples binding and catalysis

Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to their cognate tRNAs. Specific aminoacylation is dictated by a set of recognition elements that mark tRNA molecules as substrates for particular synthetases. Escherichia coli prolyl-tRNA synthetase (ProRS) has previously been shown to recognize specific bases of tRNA(Pro) in both the anticodon domain, which mediate initial complex formation, and in the acceptor stem, which is proximal to the site of catalysis. In this work, we unambiguously define the molecular interaction between E. coli ProRS and the acceptor stem of cognate tRNA(Pro). Oxidative cross-linking studies using 2'-deoxy-8-oxo-7,8-dihydroguanosine-containing proline tRNAs identify a direct interaction between a critical arginine residue (R144) in the active site of E. coli ProRS and the G72 residue in the acceptor stem of tRNA(Pro). Assays conducted with motif 2 loop variants and tRNA mutants wherein specific atomic groups of G72 were deleted, are consistent with a functionally important hydrogen-bonding network between R144 and the major groove of G72. These results taken together with previous studies suggest that breaking this key contact uncouples the allosteric interaction between the anticodon domain and the aminoacylation active site, providing new insights into the communication network that governs the synthetase-tRNA interaction.     

Association of an aminoacyl-tRNA synthetase with a putative metabolic protein in archaea

Aminoacyl-tRNA synthetases are essential enzymes that catalyze attachment of amino acids to tRNAs for decoding of genetic information. In higher eukaryotes, several synthetases associate with non-synthetase proteins to form a high-molecular mass complex that may improve the efficiency of protein synthesis. This multi-synthetase complex is not found in bacteria. Here we describe the isolation of a non-synthetase protein from the archaeon Methanocaldococcus jannaschii that was copurified with prolyl-tRNA synthetase (ProRS). This protein, Mj1338, also interacts with several other tRNA synthetases and has an affinity for general tRNA, suggesting the possibility of forming a multi-synthetase complex. However, unlike the non-synthetase proteins in the eukaryotic complex, the protein Mj1338 is predicted to be a metabolic protein, related to members of the family of H(2)-forming N(5),N(10)-methylene tetrahydromethanopterin (5,10-CH(2)-H(4)MP) dehydrogenases that are involved in the one-carbon metabolism of the archaeon. The association of Mj1338 with ProRS, and with other components of the protein synthesis machinery, thus suggests the possibility of a closer link between metabolism and decoding in archaea than in eukarya or bacteria.     

Evolutionary coadaptation of the motif 2--acceptor stem interaction in the class II prolyl-tRNA synthetase system

Known crystal structures of class II aminoacyl-tRNA synthetases complexed to their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta-strands of motif 2 located within the catalytic core of class II synthetases. To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop. We identified an arginine residue (R144) that was essential for tRNA aminoacylation but played no role in amino acid activation. Cross-linking experiments confirmed that the end of the tRNA(Pro) acceptor stem is proximal to this motif 2 loop residue. Previous work had shown that the tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved among bacteria) are important recognition elements for E. coli ProRS. We carried out atomic group "mutagenesis" studies at these two positions of E. coli tRNA(Pro) and determined that major groove functional groups at A73 and G72 are critical for recognition by ProRS. Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme. An analysis of chimeric tRNA(Pro) constructs showed that, in addition to A73 and G72, transplantation of the E. coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase. In contrast to the bacterial system, base-specific acceptor stem recognition does not appear to be used by human ProRS. Alanine-scanning mutagenesis revealed that motif 2 loop residues are not critical for tRNA aminoacylation activity of the human enzyme. Taken together, our results illustrate how synthetases and tRNAs have coadapted to changes in protein-acceptor stem recognition through evolution.     

Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. To prevent errors in protein synthesis, many synthetases have evolved editing pathways by which misactivated amino acids (pre-transfer editing) and misacylated tRNAs (post-transfer editing) are hydrolyzed. Previous studies have shown that class II prolyl-tRNA synthetase (ProRS) possesses both pre- and post-transfer editing functions against noncognate alanine. To assess the relative contributions of pre- and post-transfer editing, presented herein are kinetic studies of an Escherichia coli ProRS mutant in which post-transfer editing is selectively inactivated, effectively isolating the pre-transfer editing pathway. When post-transfer editing is abolished, substantial levels of alanine mischarging are observed under saturating amino acid conditions, indicating that pre-transfer editing alone cannot prevent the formation of Ala-tRNA Pro. Steady-state kinetic parameters for aminoacylation measured under these conditions reveal that the preference for proline over alanine is 2000-fold, which is well within the regime where editing is required. Simultaneous measurement of AMP and Ala-tRNA Pro formation in the presence of tRNA Pro suggested that misactivated alanine is efficiently transferred to tRNA to form the mischarged product. In the absence of tRNA, enzyme-catalyzed Ala-AMP hydrolysis is the dominant form of editing, with "selective release" of noncognate adenylate from the active site constituting a minor pathway. Studies with human and Methanococcus jannaschii ProRS, which lack a post-transfer editing domain, suggest that enzymatic pre-transfer editing occurs within the aminoacylation active site. Taken together, the results reported herein illustrate how both pre- and post-transfer editing pathways work in concert to ensure accurate aminoacylation by ProRS.     

Functional dissection of the eukaryotic-specific tRNA-interacting factor of lysyl-tRNA synthetase

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, referred to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNA-binding site. Their mutation into alanine led to a reduced affinity for tRNA(3)(Lys) or minimalized tRNA mimicking the acceptor-TPsiC stem-loop of tRNA(3)(Lys) and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA(Lys) networking during translation or to convey tRNA(3)(Lys) into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNA-interacting factor.