Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.     

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.     

Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.     

Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.     

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.     

Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.     

Translational incorporation of S-nitrosohomocysteine into protein

The non-protein amino acid homocysteine (Hcy), owing to its structural similarity to the protein amino acids methionine, isoleucine, and leucine, enters first steps of protein synthesis and is activated by methionyl-, isoleucyl-, and leucyl-tRNA synthetases in vivo. However, translational incorporation of Hcy into protein is prevented by editing mechanisms of these synthetases, which convert misactivated Hcy into thiolactone. The lack of efficient interactions of the side chain of Hcy with the specificity subsite of the synthetic/editing active site is a prerequisite for editing of Hcy. Thus, if the side chain thiol of Hcy were reversibly modified with a small molecule that would enhance its binding to the specificity subsite and prevent editing, such modified Hcy is predicted to be transferred to tRNA and incorporated translationally into protein. Here I show that S-nitroso-Hcy is in fact transferred to tRNA by methionyl-tRNA synthetase and incorporated into protein by the bacterium Escherichia coli. S-Nitroso-Hcy-tRNA also supports translation of mRNAs in a rabbit reticulocyte system. Removal of the nitroso group yields Hcy-tRNA and protein containing Hcy in peptide bonds. S-Nitrosylation-mediated translational incorporation of Hcy into protein may occur under natural conditions in cells and contribute to Hcy-induced pathogenesis in atherosclerosis.     

Quality control in tRNA charging -- editing of homocysteine

All living organisms conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from a gene to 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. When differences in binding energies of amino acids to an aminoacyl-tRNA synthetase are inadequate, editing is used as a major determinant of enzyme selectivity. Some incorrect amino acids are edited at the active site before the transfer to tRNA (pre-transfer editing), while others are edited after transfer to tRNA at a separate editing site (post-transfer editing). Access of natural non-protein amino acids, such as homocysteine, homoserine, or ornithine to the genetic code is prevented by the editing function of aminoacyl-tRNA synthetases. Disabling editing function leads to tRNA mischarging errors and incorporation of incorrect amino acids into protein, which is detrimental to cell homeostasis and inhibits growth. Continuous homocysteine editing by methionyl-tRNA synthetase, resulting in the synthesis of homocysteine thiolactone, is part of the process of tRNA aminoacylation in living organisms, from bacteria to man. Excessive homocysteine thiolactone synthesis in hyperhomocysteinemia caused by genetic or nutritional deficiencies is linked to human vascular and neurological diseases.     

A promiscuous aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine homologs into proteins

A mutant Escherichia coli leucyl-tRNA synthetase has been evolved for the selective incorporation of the methionine homolog 1 into proteins in yeast. This single aminoacyl-tRNA synthetase is capable of charging an amber suppressor EctRNA(CUA)(Leu) with at least eight different amino acids including methionine and cysteine homologs, as well as straight chain aliphatic amino acids. In addition we show that incorporation yields for these amino acids can be increased substantially by mutations in the editing CP1 domain of the E. coli leucyl-tRNA synthetase.     

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

Coordination of tRNA synthetase active sites for chemical fidelity

Statistical proteomes that are naturally occurring can result from mechanisms involving aminoacyl-tRNA synthetases (aaRSs) with inactivated hydrolytic editing active sites. In one case, Mycoplasma mobile leucyl-tRNA synthetase (LeuRS) is uniquely missing its entire amino acid editing domain, called CP1, which is otherwise present in all known LeuRSs and also isoleucyl- and valyl-tRNA synthetases. This hydrolytic CP1 domain was fused to a synthetic core composed of a Rossmann ATP-binding fold. The fusion event splits the primary structure of the Rossmann fold into two halves. Hybrid LeuRS chimeras using M. mobile LeuRS as a scaffold were constructed to investigate the evolutionary protein:protein fusion of the CP1 editing domain to the Rossmann fold domain that is ubiquitously found in kinases and dehydrogenases, in addition to class I aaRSs. Significantly, these results determined that the modular construction of aaRSs and their adaptation to accommodate more stringent amino acid specificities included CP1-dependent distal effects on amino acid discrimination in the synthetic core. As increasingly sophisticated protein synthesis machinery evolved, the addition of the CP1 domain increased specificity in the synthetic site, as well as provided a hydrolytic editing site.     

The Yin and Yang of tRNA: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation

Faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer RNAs (tRNAs) catalyzed by aminoacyl-tRNA synthetases. The fidelity of leucyl-tRNA synthetase (LeuRS) depends mainly on proofreading at the pre- and post-transfer levels. During the catalytic cycle, the tRNA CCA-tail shuttles between the synthetic and editing domains to accomplish the aminoacylation and editing reactions. Previously, we showed that the Y330D mutation of Escherichia coli LeuRS, which blocks the entry of the tRNA CCA-tail into the connective polypeptide 1domain, abolishes both tRNA-dependent pre- and post-transfer editing. In this study, we identified the counterpart substitutions, which constrain the tRNA acceptor stem binding within the synthetic active site. These mutations negatively impact the tRNA charging activity while retaining the capacity to activate the amino acid. Interestingly, the mutated LeuRSs exhibit increased global editing activity in the presence of a non-cognate amino acid. We used a reaction mimicking post-transfer editing to show that these mutations decrease post-transfer editing owing to reduced tRNA aminoacylation activity. This implied that the increased editing activity originates from tRNA-dependent pre-transfer editing. These results, together with our previous work, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the aminoacylation and editing activities of LeuRS.     

与人类疾病相关的几种线粒体氨基酰-tRNA合成酶

氨基酰-tRNA合成酶是一类古老的蛋白质,催化蛋白质生物合成中的第一步反应．已经发现氨基酰-tRNA合成酶还参与大量的其他生命过程,如编校、tRNA的成熟与转运、RNA的剪切、细胞因子等功能．最近的研究结果表明,线粒体氨基酰-tRNA合成酶与人类的疾病密切相关．人线粒体精氨酰-tRNA合成酶基因2号内含子中的一个单点突变导致该基因的转录本被异常剪接,造成脑桥小脑发育不全．人线粒体天冬氨酰-tRNA合成酶基因上的一系列突变致使其mRNA被快速降解或者蛋白质氨基酸一级结构的改变,导致脑干脊髓白质病变及乳糖增高症．人线粒体亮氨酰-tRNA合成酶基因的一个单核苷酸多态性与2型糖尿病密切相关．这些研究结果进一步增强了我们对于氨基酰-tRNA合成酶的生物学功能的认识,并将促进对由线粒体氨基酰-tRNA合成酶所引起线粒体病的致病机理以及治疗方法的研究．     

Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis.

Misactivation of amino acids by aminoacyl-tRNA synthetases can lead to significant errors in protein synthesis that are prevented by editing reactions. As an example, discrete sites in isoleucyl-tRNA synthetase for amino acid activation and editing are about 25 A apart. The details of how misactivated valine is translocated from one site to the other are unknown. Here, we present a kinetic study in which a fluorescent probe is used to monitor translocation of misactivated valine from the active site to the editing site. Isoleucine-specific tRNA, and not other tRNAs, is essential for translocation of misactivated valine. Misactivation and translocation occur on the same enzyme molecule, with translocation being rate limiting for editing. These results illustrate a remarkable capacity for a specific tRNA to enhance amino acid fine structure recognition by triggering a unimolecular translocation event.     

Expression and characterization of the human mitochondrial leucyl-tRNA synthetase

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.     

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.     

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre- and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2' or 3' hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.     

Mutational separation of two pathways for editing by a class I tRNA synthetase

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.     

The 2 A crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue

Leucyl-, isoleucyl- and valyl-tRNA synthetases are closely related large monomeric class I synthetases. Each contains a homologous insertion domain of approximately 200 residues, which is thought to permit them to hydrolyse ('edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid. We describe the first crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A resolution. The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure. This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional flexibly inserted domain. Comparison of native enzyme and complexes with leucine and a leucyl- adenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes significant conformational changes in the active site required for amino acid activation and tight binding of the adenylate. These changes are propagated to more distant regions of the enzyme, leading to a significantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.     

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.