Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNALeu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNALeu (hctRNALeu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNALeu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution

Multiple Quality Control Pathways Limit Non-Protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNAPhe. We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNAPhe with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNAPhe species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress

Nucleic Acids Res

Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNAThr2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNAThr1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNAThrs during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNAThr2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNAThr1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNAThr1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core

Crucial role of the Cterminal domain of Mycobacterium tuberculosis leucyl-tRNA synthetase in aminoacylation and editing

ABSTRACT The C-terminal extension of prokaryotic leucy

Aminoacyl-tRNA Synthetases

The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology

Nucleic Acids Res

Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA(76) end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA(76) accepting end of tRNA(Leu) during the aminoacylation and editing reactions. The analysis showed that the terminal A(76) is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C(74) and C(75) selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA(76) binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA(76) homogeneity of tRNAs

A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium </it>parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of <it>Plasmodium </it>genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (<it>aaRS</it>s) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the <it>Plasmodium falciparum </it>genome to identify and classify <it>aaRSs </it>in this organism.</p> <p>Results</p> <p>Using various computational and bioinformatics tools, we have identified 37 <it>aaRS</it>s in <it>P. falciparum</it>. Our key observations are: (i) fraction of proteome dedicated to <it>aaRS</it>s in <it>P. falciparum </it>is very high compared to many other organisms; (ii) 23 out of 37 <it>Pf-aaRS </it>sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of <it>Pf-aaRSs </it>vary considerably at various life cycle stages of the parasite; (iv) several <it>PfaaRSs </it>posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite <it>aaRS</it>s to bacterial and plants <it>aaRSs</it>; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite <it>aaRSs</it>.</p> <p>Conclusion</p> <p>We have identified 37 <it>Pf-aaRSs </it>based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 <it>Pf-aaRSs </it>based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.</p

Mechanisms of leucyl-tRNA synthetase dependent group I intron splicing

Leucyl-tRNA synthetase (LeuRS) plays dual roles within the yeast mitochondria. In addition to protein synthesis, it is also essential to RNA splicing of critical respiratory genes. The LeuRS collaborates with a maturase to excise the bI4 and aI4α introns from the cob and cox1α genes respectively. The LeuRS-based suppressor mutations have been isolated within the amino acid editing CP1 domain and restore native RNA splicing activity in the presence of an inactive maturase. Mutational analysis of these sites and the regions that surround them demonstrated that certain substitutions can also inactivate LeuRS-dependent splicing activity under in vivo and in vitro conditions. Binding measurements suggest that these suppressor sites are important in maintaining interaction between LeuRS and the group I intron RNA. Thus, CP1 domain binds specifically to the bI4 and aI4α intron to promote RNA splicing. In addition to LeuRS from yeast mitochondria (ymLeuRS), diverse LeuRSs from varied origins such as M. tuberculosis and human mitochondria complement the ymLeuRS activities. Similarly, wild-type E. coli LeuRS (EcLeuRS) complemented a ymLeuRS null strain. Interestingly, at reduced levels of EcLeuRS expression in yeast mitochondria, the heterologous synthetase supported protein synthesis, but not intron splicing. Thus, it is a weak splicing suppressor. Surprisingly, a gain of splicing activity was exhibited by positive charge substitutions at the Ala293 position, suggesting that this Ala293 can be adapted for alternative activities. Preliminary footprinting data suggest that LeuRS binds to the P4-P6 core region of the bI4 intron that is cognate to LeuRS. The RNA duplex mimics of the P6 helix were designed and it was shown that LeuRS promotes their annealing in an ATP-independent manner. Domain analysis of LeuRS shows that the C-terminal domain is critical to the RNA annealing activity. Yeast mitochondrial tRNALeu (ymtRNALeu) competitively inhibit annealing. Also, an ymtRNALeu variable-stem-like region was identified on the P6 stem that is important for LeuRS-dependent annealing. These data support that the annealing and tRNA variable arm binding sites overlap on the C-terminal domain of LeuRS. It was shown that the overhang location and length of the duplexes are important features that LeuRS recognizes. It was hypothesized that LeuRS plays a key role in remodeling specific group I intron ribozymes so that they can productively self-splice

Editing by leucyl-trna synthetase: Discrimination of norvaline and isoleucine

Aminoacyl tRNA-synthetases (AARS) are housekeeping enzymes that are tasked with accurate synthesis of aminoacylated tRNA for protein synthesis and other cellular functions. The specificity of amino acid attachment challenges the AARSs that need to distinguish between structurally similar amino acids. In such cases, AARSs have developed editing mechanisms to circumvent the issue of misaminoacylation. Leucyl-tRNA synthetase (LeuRS), for instance selectively edits misactivated and mischarged non-leucine amino acids via pre-transfer editing of misactivated adenylates in the synthetic site or by hydrolyzing mischarged amino acids in the CP1 editing domain. The enzyme’s dependence between the two editing mechanisms can shift based on the origin from which the AARS is derived, the amino acid that is targeted for editing, or presence of a mutation in the enzyme. In the absence of the CP1 domain, E. coli LeuRS (LeuRS-ΔCP1) maintains fidelity by clearing non-leucine aminoacyl-adenylates in the enzyme’s synthetic site. The intact tRNA 3’-terminal adenosine (A76) residue is a prerequisite for aminoacylation. Leveraging A76 essentiality tRNA analogues were designed to investigate amino acid dependent specificity of editing by LeuRS. The tRNA analogues were synthesized by addition of a modified adenosine triphosphate to an in vitro transcribed E. coli tRNALeuUAA using the CCA-adding enzyme from E. coli. Incorporation of unchargeable tRNA analogues stimulated ATP hydrolysis by wild type LeuRS in the presence of norvaline. In contrast, pre-transfer editing occurs independent of the tRNA for LeuRS-ΔCP1, which lacks the CP1 domain. Therefore it is hypothesized that the CP1 domain of LeuRS plays a critical role for tRNA-dependent pre-transfer editing

인간 세포질 aminoacyl-tRNA synthetases의 동적인 상호작용과 효소 활성에 대한 분석법 개발

학위논문 (박사) -- 서울대학교 대학원 : 약학대학 약학과, 2020. 8. 김성훈.To the best of our knowledge, protein synthesis (translation) is a universal process, which resides in all extant lifeforms. An aminoacyl-tRNA synthetase (ARS) takes a role in the very first step of the translation process; it catalyzes esterification (aminoacylation) of a specific amino acid on its cognate transfer RNAs (tRNAs) to make aminoacylated tRNAs (aminoacyl-tRNAs). The aminoacyl-tRNA delivers the amino acid to the ribosome which catalyzes the translation of a messenger RNA (mRNA) into a polypeptide chain. The cytoplasmic ARSs are differentially regulated in different species; they have gained additional domains and noncanonical functions throughout evolution, and the largest multi-tRNA synthetase complex (MSC) among the eukaryotes exists in higher eukaryotes, which is comprised of eight ARSs for eight or nine amino acids. Among them, the mammalian MSC is the most complexed one, which is composed of eight cytoplasmic ARSs for nine amino acids, and three scaffold proteins. Consequently, nearly half of the aminoacyl-tRNA efflux becomes concentrated at the MSC. Stable supply of the aminoacyl-tRNA to the ribosome is, therefore, considered to be a major role of the mammalian MSC. Furthermore, the mammalian MSC also serves as a reservoir for releasable ARSs or scaffold proteins to support the noncanonical functions of them. In part I, a split-luciferase complementation system was applied to investigate the configuration of the MSC in live mammalian cells. Multiplex interconnections between the components of the MSC were simplified into binary protein-protein interactions, and pairwise comparison of the interactions reconstituted a framework that is consistent with previous in vitro studies. Reversibility of the split-luciferase reporter binding demonstrated convertible organization of the mammalian MSC, including interferon gamma (IFNγ)-stimulated glutamyl-prolyl-tRNA synthetase 1 (EPRS1) release, as well as the cooperation with the ribosome bridged by the tRNAs. The cell-based analysis provided an improved understanding of the flexible framework of the mammalian MSC in physiological conditions. On the other hand, abnormality of the aminoacylation has been implicated in a wide variety of cancer pathologies. The ARSs exist in large excess in cancer cells due to their increased demand for the protein synthesis. Meanwhile, most other translation apparatuses are quantitatively limited. There has been no report for mutations of the ARSs that demonstrate constitutive activity of the aminoacylation; the hyperactivity of the ARSs may disrupt stable association of the MSC. Hence, interference of the aminoacylation activity is expected to be independent of genotype variation and may not develop drug resistance. In part II, a high-throughput screen (HTS) platform was established to find the mammalian ARS inhibitors. The ARSs of rabbit reticulocyte closely resemble both the individual and complexed structures of human ones. Therefore, an in vitro translation system with the rabbit-reticulocyte lysate may predispose active compounds to be readily applicable for mankind. The assay was further validated for identifying familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for the purpose. Having demonstrated excellent quality control (QC) parameters and reproducibility, it is proven ready for further HTS campaign with large molecular entities.단백질 합성 (번역)은 모든 형태의 생명이 가지고 있는 공통적인 특성이다. 아미노산-운반RNA 연결효소 (aminoacyl-tRNA synthetase; ARS)는 단백질 합성 과정에서 가장 첫 번째 단계를 담당하고 있다. 아미노산-운반RNA 연결효소는 운반RNA (transfer RNA; tRNA)와 상보적인 아미노산 (amino acid) 사이의 에스터화 반응 (esterification/aminoacylation)을 촉매하여 아미노산-운반RNA 중합체 (aminoacyl-tRNA)로 연결한다. 생성된 아미노산-운반RNA 중합체는 리보솜 (ribosome)으로 전달되어 전령RNA (messenger RNA; mRNA)를 펩타이트 중합체 (polypeptide)로 번역하는 과정의 재료로 사용된다. 세포질 아미노산-운반RNA 연결효소 (cytoplasmic ARS)는 종에 따라 세포내에서 제어되는 방식이 다르다. 이 효소들은 진화과정 동안 추가적인 단백질 도메인 (protein domain)과 새로운 기능들을 획득해왔다. 또한 고등 진핵생물 (higher eukaryote)에는 진핵생물 (eukaryote) 중에서 가장 큰 아미노산-운반RNA 연결효소 복합체 (multi-tRNA synthetase complex; MSC)가 존재하며, 이 복합체는 8종류의 아미노산-운반RNA 연결효소가 8 내지는 9종류의 아미노산을 담당한다. 이 중에서 포유동물의 아미노산-운반RNA 연결효소 복합체가 가장 복잡한 형태인데, 8종류의 세포질 아미노산-운반RNA 연결효소가 9종류의 아미노산을 담당하며, 추가적으로 3종류의 뼈대 단백질 (scaffold protein)이 존재한다. 이처럼 포유동물의 세포 내에서는 아미노산-운반RNA 중합체 이동의 절반 정도가 아미노산-운반RNA 연결효소 복합체에서 시작되고 있다. 따라서 아미노산-운반RNA 중합체를 리보솜으로 안정적으로 공급하는 것이 포유동물 아미노산-운반RNA 연결효소 복합체의 가장 중요한 역할 중의 하나일 것이라 예상된다. 한편으로, 몇몇의 세포질 아미노산-운반RNA 연결효소들과 뼈대 단백질들은 복합체에서 벗어나 새로운 기능을 수행하기 때문에, 아미노산-운반RNA 연결효소 복합체는 이들을 위한 저장고 (reservoir)가 되기도 한다. 본 논문의 첫 번째 부분에서는 분할 루시퍼레이즈 상보 시스템 (split-luciferase complementation system)을 사용하여 포유동물 세포 내에서 아미노산-운반RNA 연결효소 복합체의 구성을 탐색하였다. 구성요소들 간의 복합적인 상호연결은 두 단백질 간의 상호작용 (binary protein-protein interaction; binary PPI)의 합으로 단순화시켰고, 그들간의 쌍별 비교 (pairwise comparison; 구성 요소들의 서로 다른 모든 조합을 비교하는 방식)를 통하여 기존의 생체 외 연구 (in vitro studies)에 상응하는 복합체의 골조 (framework)를 유추해낼 수 있었다. 그리고 분할 루시퍼레이즈 리포터 (split-luciferase reporter) 간의 결합이 가역적이라는 점을 이용하여 인터페론감마 (interferon gamma; IFNγ)에 의한 글루타민-프롤린-운반RNA 연결효소 (glutamyl-prolyl-tRNA synthetase 1; EPRS1)의 방출이나, 운반RNA를 매개로 한 리보솜과 아미노산-운반RNA 연결효소 복합체의 협업과 같은 여러 자극들에 의한 복합체 내의 역동적인 구조 변화를 관찰하였다. 본 연구는 이와 같이 세포를 기반으로 한 분석법을 바탕으로 생리적 환경 (physiological condition)에서 포유동물 아미노산-운반RNA 연결효소 복합체의 골조가 유동적으로 변화하고 있음을 밝혀낼 수 있었다. 한편, 정상적이지 못한 아미노산-운반RNA 연결반응의 존재는 여러 종류의 암에서 잘 알려져 있다. 아미노산-운반RNA 연결효소는 암세포에서 과량으로 존재하며, 암이 진행되면서 늘어난 단백질 합성 요구량을 충족시킨다. 이는 대부분의 다른 단백질 합성 요소들이 암세포에서 과량으로 존재하지 않는다는 사실과 대비된다. 또한 아미노산-운반RNA 연결효소의 활성을 지속적으로 유지시키는 돌연변이는 지금까지 알려져 있지 않다. 이는 효소활성이 비정상적으로 높아진 아미노산-운반RNA 연결효소는 아미노산-운반RNA 연결효소 복합체의 형성과 유지를 저해하기 때문일 것이다. 따라서 아미노산-운반RNA 연결반응을 저해하는 치료법은 환자 개개인의 유전체 다양성 (genotype variation)에 상관없이 효과를 낼 수 있고, 약물 저항성도 나타나지 않을 것이라 기대된다. 본 논문의 두 번째 부분에서는 고속 대량 스크리닝 플랫폼 (high-throughput screening platform)을 구축하여 포유동물 아미노산-운반RNA 연결효소의 저해제를 찾고자 하였다. 이 시스템에서 사용된 토끼의 망상적혈구 용해물 (rabbit-reticulocyte lysate) 내의 아미노산-운반RNA 연결효소들은 단독 또는 결합 구조가 인간의 효소나 그 복합체와 매우 가깝게 닮아있기 때문에 찾아낸 화합물이 인간에게 바로 적용될 수 있는 가능성을 높여준다. 이 시스템은 본 연구에서 수행된 선행 스크리닝 (pilot screening)에서 에메틴 (emetine)과 같이 잘 알려진 단백질 합성 저해제를 찾아내었을 뿐만 아니라, 훌륭한 품질관리 매개변수 (quality control parameters; QC parameters)와 결과의 반복성을 보여주었다. 따라서 이 시스템은 추후 대량 화합물 라이브러리를 타겟으로 한 고속 대량 스크리닝에 활용이 용이할 것으로 기대된다.Introduction 1 Part I. Cell-based analysis of pairwise interactions between the components of the multi-tRNA synthetase complex 11 Graphical abstract 11 Highlights 12 Introduction 13 Materials and methods 18 Results 24 Discussion 34 Figures and table 37 Part II. High-throughput screen for protein synthesis inhibitors targeting aminoacyl-tRNA synthetases 89 Graphical abstract 89 Highlights 90 Introduction 91 Materials and methods 95 Results 98 Discussion 106 Figures and tables 107 Glossary 123 Abbreviations 124 References 128 국문초록 150Docto