Regulation of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The regulation of synthesis of valyl-, leucyl-, and isoleucyl-transfer ribonucleic acid (tRNA) synthetases was examined in strains of Escherichia coli and Salmonella typhimurium. When valine and isoleucine were limiting growth, the rate of formation of valyl-tRNA synthetase was derepressed about sixfold; addition of these amino acids caused repression of synthesis of this enzyme. The rate of synthesis of the isoleucyl- and leucyl-tRNA synthetases was derepressed only during growth restriction by the cognate amino acid. Restoration of the respective amino acid to these derepressed cultures caused repression of synthesis of the aminoacyl-tRNA synthetase, despite the resumption of the wild-type growth rate.     

On the Classes of Aminoacyl-tRNA Synthetases,Amino Acids and the Genetic Code

The division of the aminoacyl-tRNA synthetases in two classes is compared with a division of the amino acids in two classes, obtained from the AAIndex databank by a principal component analysis. The division of the enzymes in Classes I and II follows to a great extent a division in the chemical and biological properties of their cognate amino acids. Furthermore, the phylogenetic trees of Classes I and II enzymes are highly correlated with dendrograms obtained for their cognate amino acids by using the indices in the AAIndex database. We argue that the evolution of aminoacyl-tRNA synthetases was determined by the characteristics of their corresponding amino acids. We interpret these results considering models for the origin and evolution of the genetic code in which an initial version, containing fewer amino acids, was modified by the incorporation of new amino acids following duplication and divergence of previous synthetases and tRNA molecules.     

Studies on the regulation of the branched chain amino acyl-tRNA synthetases of the fungusNeurospora crassa

Summary The specific activities of the branched chain amino acyl-tRNA synthetases from the cytosolic and mitochondrial fractions ofN. crassa were low in dormant conidia and increased during germination, reaching a maximum 8 h after inoculation. This stage of development is characterised by high rates of many other cellular activities.The increases in activity of synthetases of both cytosol and mitochondria are inhibited by cycloheximide indicating that they are synthesized on cytoplasmic ribosomes. The mitochondrial synthetases show a stimulation of their specific activity when mitochondrial RNA and protein synthesis are inhibited by either ethidium bromide or chloramphenicol suggesting that a mitochondrial translation product regulates the synthesis of the mitochondrial synthetases.The activities of amino acyl-tRNA synthetases are dependent on energy production. When respiration is uncoupled from oxidative phosphorylation, synthetase specific activities decrease although the activities of other mitochondrial enzymes like NADH-dehydrogenase increase. This phenomenon suggests that more than one mechanism regulates the synthesis of mitochondrial proteins which are formed on cytoplasmic ribosomes.The synthesis of branched chain amino acyl-tRNA synthetases ofNeurospora is neither repressed by their cognate amino acids, nor is there inhibition by the precursors of these amino acids, as has been observed in other amino acyl-tRNA synthetases of various organism includingNeurospora.     

氨酰-tRNA合成酶的研究进展

氨酰-tRNA合成酶催化特异的氨基酸与同源tRNA氨酰化,从而保证了遗传密码翻译的忠实性。这些古老而保守的蛋白质分子除了具有酶的功能外,在哺乳动物细胞中还发现了多种其他功能,具有重要的应用价值。在寻找具有全新作用机制的新抗生素以应对日益严重的抗生素耐药现象过程中,氨酰-tRNA合成酶是细菌蛋白质合成过程中重要的、新颖的靶标,成为关注的重点。定向突变的氨酰-tRNA合成酶可以用来定点掺入非天然氨基酸,扩展蛋白质工程。今后,随着人们对氨酰-tRNA合成酶研究的不断深入,它们还可能用来治疗肿瘤等多种疾病。     

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

Editing of errors in selection of amino acids for protein synthesis.

All living cells must conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from gene to finished protein product. One crucial "quality control" point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. During selection of amino acids, synthetases very often have to distinguish the cognate substrate from a homolog having just one fewer methyl group in its structure. The binding energy of a methyl group is estimated to contribute only a factor of 100 to the specificity of binding, yet synthetases distinguish such closely related amino acids with a discrimination factor of 10,000 to 100,000. Examples of this include methionine versus homocysteine, isoleucine versus valine, alanine versus glycine, and threonine versus serine. Many investigators have demonstrated in vitro the ability of certain aminoacyl-tRNA synthetases to edit, that is, correct or prevent incorrect attachment of amino acids to tRNA molecules. Several major editing pathways are now established from in vitro data. Further, at least some aminoacyl-tRNA synthetases have recently been shown to carry out the editing function in vivo. Editing has been demonstrated to occur in both Escherichia coli and Saccharomyces cerevisiae. Significant energy is expended by the cell for editing of misactivated amino acids, which can be reflected in the growth rate. Because of this, cellular levels of aminoacyl-tRNA synthetases, as well as amino acid biosynthetic pathways which yield competing substrates for protein synthesis, must be carefully regulated to prevent excessive editing. High-level expression of recombinant proteins imposes a strain on the biosynthetic capacity of the cell which frequently results in misincorporation of abnormal or wrong amino acids owing in part to limited editing by synthetases. Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA aminoacylation. The availability of X-ray crystallographic structures of some synthetases, combined with site-directed mutagenesis, allows insights into molecular details of the extraordinary selectivity of synthetases, including the editing function.     

A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu

Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site. Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA. Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.     

Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3' end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of post-transfer editing, or the N- or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.     

Amino acid modifications on tRNA

The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation. Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa-tRNA synthetases. However, in the case of four amino acids (Gln, Asn, Cys and Sec), aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life. The process begins with the charging of noncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNA(Cys) formation or by synthetases with relaxed specificity, such as the non-discriminating glutamyl-tRNA, non-discriminating aspartyl-tRNA and seryl-tRNA synthetases. The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA, which is catalyzed by a group of tRNA-dependent modifying enzymes, such as tRNA-dependent amidotransferases, Sep-tRNA:Cys-tRNA synthase, O-phosphoseryl-tRNA kinase and Sep-tRNA:Sec-tRNA synthase. The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.     

Modulation of substrate specificity within the amino acid editing site of leucyl-tRNA synthetase

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have evolved editing mechanisms to ensure fidelity in this first step of protein synthesis. The amino acid editing site for leucyl- (LeuRS) and isoleucyl- (IleRS) tRNA synthetases reside within homologous CP1 domains. In each case, a threonine-rich peptide and a second conserved GTG region that are separated by about 100 amino acids comprise parts of the hydrolytic editing site. While a number of sites are conserved between these two enzymes and likely confer a commonality to the mechanisms, some positions are idiosyncratic to LeuRS or IleRS. Herein, we provide evidence that a conserved arginine and threonine at respective sites in LeuRS and IleRS diverged to confer amino acid substrate recognition. This site complements other sites in the amino acid binding pocket of the editing active site of Escherichia coli LeuRS, including Thr252 and Val338, which collectively fine-tune amino acid specificity to confer fidelity.     

Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.     

Decoding the impact of disease-causing mutations in an essential aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are housekeeping enzymes that catalyze the specific attachment of amino acids onto cognate tRNAs, providing building blocks for ribosomal protein synthesis. Owing to the absolutely essential nature of these enzymes, the possibility that mutations in their sequence could be the underlying cause of diseases had not been foreseen. However, we are learning of patients bearing familial mutations in aminoacyl-tRNA synthetases at an exponential rate. In a recent issue of JBC, Jin et al. analyzed the impact of two such mutations in the very special bifunctional human glutamyl-prolyl-tRNA synthetase and convincingly decode how these mutations elicit the integrated stress response.     

Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation

Aminoacyl-tRNA synthetases attach amino acids to the 3' termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13-18, 2002) discussed new research into the structure-function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code.     

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNA^Ile and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.     

氨酰tRNA合成酶的分子网络和功能

氨酰tRNA合成酶是生命进化过程中最早出现的一类蛋白质,氨酰tRNA合成酶帮助氨基酸转移到相应的tRNA上,进而参与蛋白质的合成保证了生命体的严谨性和多样性.随着后基因组时代的到来,氨酰tRNA合成酶的结构和功能成为新的研究热点.结构生物学和生物信息学的研究结果表明,氨酰tRNA合成酶在真核生物体内以多聚复合物的形式行使功能,形成复杂的分子网络体系.最新的实验证据显示,氨酰tRNA合成酶不但是蛋白质合成过程中一类最重要的酶,而且参与了转录、翻译水平的调控、RNA剪接、信号传导和免疫应答等众多生命活动.     

氨酰tRNA 合成酶抑制剂作为新型抗感染药物的研究进展

细菌耐药性的不断上升对现有阶段的抗生素类药物提出了一个严峻的挑战,同时也掀起了针对于新靶标的抗菌药物的研究。氨酰tRNA合成酶(aaRS)催化特定氨基酸连接到相应的tRNA分子上,在蛋白质的合成过程中起着必不可少的作用。氨酰tRNA合成酶的抑制会导致蛋白质合成的停止,扰乱细菌和真菌的生长,因此氨酰tRNA合成酶是一类潜在的抗感染靶标。本文分别综述了天然产物及其衍生的aaRS抑制剂,底物和反应中间体模拟物,通过合成和通过虚拟筛选得到的aaRS抑制剂作为新型抗细菌和抗真菌药物的研究进展,并对aaRS的靶标特点、分类和催化机制作一简要介绍。     

Amino acid selectivity in the aminoacylation of coenzyme A and RNA minihelices by aminoacyl-tRNA synthetases

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TpsiC helix of tRNA(Ile) or tRNA(Val) were aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.     

The glycyl-tRNA synthetase of Chlamydia trachomatis.

Aminoacyl-tRNA synthetases specifically charge tRNAs with their cognate amino acids. A prototype for the most complex aminoacyl-tRNA synthetases is the four-subunit glycyl-tRNA synthetase from Escherichia coli, encoded by two open reading frames. We examined the glycyl-tRNA synthetase gene from Chlamydia trachomatis, a genetically isolated bacterium, and identified only a single open reading frame for the chlamydial homolog (glyQS). This is the first report of a prokaryotic glycyl-tRNA synthetase encoded by a single gene.     

An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing

Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield DeltaINS-ProRS. While DeltaINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction.