Duplication of leucyl-tRNA synthetase in an archaeal extremophile may play a role in adaptation to variable environmental conditions

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that play a fundamental role in protein synthesis. They catalyze the esterification of specific amino acids to the 3′-end of their cognate tRNAs and therefore play a pivotal role in protein synthesis. Although previous studies suggest that aaRS-dependent errors in protein synthesis can be beneficial to some microbial species, evidence that reduced aaRS fidelity can be adaptive is limited. Using bioinformatics analyses, we identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of the archaeal family Sulfolobaceae. Remarkably, one copy, designated LeuRS-I, had key amino acid substitutions within its editing domain that would be expected to disrupt hydrolytic editing of mischarged tRNALeu and to result in variation within the proteome of these extremophiles. We found that another copy, LeuRS-F, contains canonical active sites for aminoacylation and editing. Biochemical and genetic analyses of the paralogs within Sulfolobus islandicus supported the hypothesis that LeuRS-F, but not LeuRS-I, functions as an essential tRNA synthetase that accurately charges leucine to tRNALeu for protein translation. Although LeuRS-I was not essential, its expression clearly supported optimal S. islandicus growth. We conclude that LeuRS-I may have evolved to confer a selective advantage under the extreme and fluctuating environmental conditions characteristic of the volcanic hot springs in which these archaeal extremophiles reside.

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

Biochemical Journal ◽

10.1042/bj20111925 ◽

2012 ◽

Vol 443 (2) ◽

pp. 477-484 ◽

Cited By ~ 13

Author(s):

Min Tan ◽

Wei Yan ◽

Ru-Juan Liu ◽

Meng Wang ◽

Xin Chen ◽

...

Keyword(s):

Catalytic Efficiency ◽

Trna Synthetase ◽

Modular Construction ◽

Aquifex Aeolicus ◽

Trna Synthetases ◽

Naturally Occurring ◽

Aminoacyl Trna Synthetases ◽

Editing Domain ◽

Editing Activity ◽

Shed Light

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.

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Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

mSphere ◽

10.1128/mspheredirect.00340-17 ◽

2017 ◽

Vol 2 (4) ◽

Author(s):

Sanya Chadha ◽

N. Arjunreddy Mallampudi ◽

Debendra K. Mohapatra ◽

Rentala Madhubala

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Essential Gene ◽

Protozoan Parasite ◽

Protein Translation ◽

Trna Synthetase ◽

Parasite Growth ◽

Coding Sequences ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.

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Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain fromAeropyrum pernix

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309112042066 ◽

2012 ◽

Vol 68 (11) ◽

pp. 1390-1393 ◽

Cited By ~ 1

Author(s):

Sadeem Ahmad ◽

Antony S. K. Sravankumar ◽

Shobha P. Kruparani ◽

Rajan Sankaranarayanan

Keyword(s):

Trna Synthetase ◽

Asymmetric Unit ◽

Crystallographic Investigation ◽

X Ray ◽

Trna Synthetases ◽

Aeropyrum Pernix ◽

Substrate Analogues ◽

Aminoacyl Trna Synthetases ◽

Editing Domain ◽

Evolutionary Aspects

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeonPyrococcus abysiiin complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeonAeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase,i.e.ThrRS1 and ThrRS2, encoded by two different genes,thrS1andthrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 fromA. pernixis reported. The crystals belong to either theP41212 orP43212 space group and consist of one monomer per asymmetric unit.

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Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination

Biochemical Journal ◽

10.1042/bj20051249 ◽

2006 ◽

Vol 394 (2) ◽

pp. 399-407 ◽

Cited By ~ 20

Author(s):

Yunqing Liu ◽

Jing Liao ◽

Bin Zhu ◽

En-Duo Wang ◽

Jianping Ding

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Crystal Structures ◽

Active Site ◽

Binding Pocket ◽

Trna Synthetase ◽

Vital Role ◽

Common Mechanism ◽

Trna Synthetases ◽

Editing Domain

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

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Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy

Nucleic Acids Research ◽

10.1093/nar/gkab856 ◽

2021 ◽

Author(s):

Pooja Srinivas ◽

Rebecca E Steiner ◽

Ian J Pavelich ◽

Ricardo Guerrero-Ferreira ◽

Puneet Juneja ◽

...

Keyword(s):

Salmonella Enterica Serovar Typhimurium ◽

Binding Pocket ◽

Trna Synthetase ◽

Human Infection ◽

Hydroxyl Group ◽

High Fidelity ◽

Trna Synthetases ◽

Cryo Electron Microscopy ◽

Serovar Typhimurium ◽

Editing Domain

Abstract High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.

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Seryl tRNA synthetase cooperates with POT1 to regulate telomere length and cellular senescence

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-019-0078-1 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 5

Author(s):

Yingxi Li ◽

Xiyang Li ◽

Mei Cao ◽

Yuke Jiang ◽

Jie Yan ◽

...

Keyword(s):

Protein Synthesis ◽

Telomere Length ◽

Cellular Senescence ◽

Direct Interaction ◽

Causative Factor ◽

Trna Synthetase ◽

Dna Repeats ◽

Telomeric Dna ◽

Trna Synthetases ◽

Regulate Telomere Length

AbstractDeregulated telomere length is a causative factor in many physiological and pathological processes, including aging and cancer. Many studies focusing on telomeres have revealed important roles for cooperation between the Shelterin protein complex and telomerase in maintaining telomere length. However, it remains largely unknown whether and how aging-related stresses, such as deregulated protein homeostasis, impact telomere length. Here, we explored the possible roles of aminoacyl tRNA synthetases (AARSs), key enzymes catalyzing the first reactions in protein synthesis, in regulating telomere length and aging. We selected seryl tRNA synthetase (SerRS) since our previous studies discovered expanded functions of SerRS in the nucleus in addition to its canonical cytoplasmic role in protein synthesis. In this study, we revealed that overexpression of SerRS promoted cellular senescence and inhibited the growth of cervical tumor xenografts in mice by triggering the senescence of tumor cells. In the nucleus, SerRS directly bound to telomeric DNA repeats and tethered more POT1 proteins to telomeres through a direct interaction between the UNE-S domain of SerRS and the OB1 domain of POT1. We further demonstrated that SerRS-induced enrichment of POT1 prevented the recruitment of telomerase to telomeres, resulting in progressive telomere shortening. Our data suggested a possible molecular link between protein synthesis and telomere length control, the deregulation of which may be associated with aging and cancer.

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Nucleolar Localization of Human Methionyl–Trna Synthetase and Its Role in Ribosomal RNA Synthesis

The Journal of Cell Biology ◽

10.1083/jcb.149.3.567 ◽

2000 ◽

Vol 149 (3) ◽

pp. 567-574 ◽

Cited By ~ 77

Author(s):

Young-Gyu Ko ◽

Young-Sun Kang ◽

Eun-Kyoung Kim ◽

Sang Gyu Park ◽

Sunghoon Kim

Keyword(s):

Protein Synthesis ◽

Ribosomal Rna ◽

Rna Synthesis ◽

Trna Synthetase ◽

Nucleolar Localization ◽

Trna Synthetases ◽

Quiescent Cells ◽

Aminoacyl Trna Synthetases ◽

Polymerase I ◽

Methionyl Trna Synthetase

Human aminoacyl–tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl–tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.

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Evolution of Leucyl-tRNA Synthetase Through Eukaryotic Speciation

American Journal of Undergraduate Research ◽

10.33697/ajur.2017.024 ◽

2017 ◽

Vol 14 (3) ◽

Cited By ~ 1

Author(s):

Katelyn Unvert ◽

Frank Kovacs ◽

Chi Zhang ◽

Rachel Hellmann-Whitaker ◽

Katelin Arndt

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Active Sites ◽

Sequence Data ◽

Research Effort ◽

Trna Synthetase ◽

Trna Synthetases ◽

Speciation Process ◽

Evolutionary Relatedness ◽

Vertical Gene Transfer

Aminoacyl-tRNA synthetases (aaRSs) are part of the cellular translation machinery and as such, they are essential enzymes for every known cell. Due to their ubiquitous nature, their evolutionary history has been intensely researched to better understand the origins of life on a molecular level. Herein, we examine the evolutionary relatedness of leucyl-tRNA synthetases (LeuRS) from each major eukaryotic branch through the speciation process. This research effort was centered on amino acid sequence data as well as generating homology protein models for each LeuRS enzyme. Comparative analysis of this sequence and structural data for LeuRS amongst eukaryotes has indicated a high level of conservation within the active sites of these enzymes. Phylogenetic analysis confirmed this high degree of conservation as well as established evolutionary relatedness between these LeuRS enzymes. Based on this data, vertical gene transfer propagated LeuRS throughout the eukaryotic domain. Horizontal gene transfer and domain acquisition events were not observed within the eukaryotic organisms studied. Our data also highlighted LeuRS adaptation through the speciation process due to slight variability of scaffolding residues outside of the active site regions. We hypothesize that this variability may be due to mechanistic differences amongst LeuRS enzymes that have assumed non-translational functionality through the evolutionary process. KEYWORDS: tRNA Synthetase; Leucyl-tRNA Synthetase; Eukaryotic Evolution; LeuRS Conservation; Vertical Gene Transfer; Horizontal Gene Transfer; Convergent Evolution; Primordial Enzymes

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Aminoacyl tRNA Synthetases as Malarial Drug Targets: A Comparative Bioinformatics Study

10.1101/440891 ◽

2018 ◽

Author(s):

Dorothy Wavinya Nyamai ◽

Özlem Tastan Bishop

Keyword(s):

Amino Acid ◽

Drug Targets ◽

Motif Discovery ◽

Protein Translation ◽

Trna Synthetase ◽

Potential Drug ◽

Multiple Sequence ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Potential Drug Targets

AbstractTreatment of parasitic diseases has been challenging due to the development of drug resistance by parasites, and thus there is need to identify new class of drugs and drug targets. Protein translation is important for survival of plasmodium and the pathway is present in all the life cycle stages of the plasmodium parasite. Aminoacyl tRNA synthetases are primary enzymes in protein translation as they catalyse the first reaction where an amino acid is added to the cognate tRNA. Currently, there is limited research on comparative studies of aminoacyl tRNA synthetases as potential drug targets. The aim of this study is to understand differences between plasmodium and human aminoacyl tRNA synthetases through bioinformatics analysis. Plasmodium falciparum, P. fragile, P. vivax, P. ovale, P. knowlesi, P. bergei, P. malariae and human aminoacyl tRNA synthetase sequences were retrieved from UniProt database and grouped into 20 families based on amino acid specificity. Despite functional and structural conservation, multiple sequence analysis, motif discovery, pairwise sequence identity calculations and molecular phylogenetic analysis showed striking differences between parasite and human proteins. Prediction of alternate binding sites revealed potential druggable sites in PfArgRS, PfMetRS and PfProRS at regions that were weakly conserved when compared to the human homologues. These differences provide a basis for further exploration of plasmodium aminoacyl tRNA synthetases as potential drug targets.

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Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code

eLife ◽

10.7554/elife.02501 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 56

Author(s):

Tammy J Bullwinkle ◽

Noah M Reynolds ◽

Medha Raina ◽

Adil Moghal ◽

Eleftheria Matsa ◽

...

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Genetic Code ◽

Trna Synthetase ◽

Control Mechanisms ◽

Cellular Toxicity ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

The Cost

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

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