Mammalian prolyl-tRNA synthetase corresponds to the approximately 150 kDa subunit of the high-M(r) aminoacyl-tRNA synthetase complex.

The high-M(r) aminoacyl-tRNA synthetase complex previously purified from sheep liver differed from those isolated from several other mammalian sources by the absence of prolyl-tRNA synthetase activity and the presence of glutamyl tRNA synthetase as a polypeptide of 85 kDa instead of 150 kDa. Using a milder extraction procedure that minimizes proteolysis, we now report the isolation of a sheep liver complex that contains both prolyl-tRNA synthetase activity and the 150-kDa polypeptide. The correspondence between prolyl-tRNA synthetase and the 150-kDa polypeptide, inferred from the results of several approaches reported in this study, was further demonstrated by showing that antibodies to a free form of sheep liver prolyl-tRNA synthetase generated by endogenous proteolysis, specifically reacted with the 150-kDa components of the complexes from sheep and rabbit, but failed to react with the previously purified complex from sheep that contained neither prolyl-tRNA synthetases activity nor the 150-kDa component. Moreover, we show that the 150-kDa polypeptide is also recognized by antibodies to the 85-kDa polypeptide previously assigned to glutamyl-tRNA synthetase. The possibility that the largest subunit of the mammalian high-M(r) complexes may be a bifunctional protein encoding both glutamyl- and prolyl-tRNA synthetase activities is considered and discussed in light of the recently published sequence of the corresponding polypeptide from HeLa cells. In accordance with this prediction, we show that the amino acid sequence of the carboxyl-terminal moiety of this bifunctional polypeptide shows significant similarity to the sequence of prolyl-tRNA synthetase from Escherichia coli.     

家蚕氨酰-tRNA合成酶基因分析

为了探讨家蚕氨酰-tRNA合成酶(BmaaRS)基因的数目、种类、结构及起源, 利用家蚕基因组数据和EST数据进行了BmaaRS基因的电子克隆, 结果表明, 家蚕核基因组中含有2套不同的aaRS核基因, 分别编码线粒体和细胞质BmaaRS, 但编码线粒体BmSerRS的基因有2个, 可能缺少编码细胞质的BmHisRS基因和编码线粒体的BmGlnRS、BmLysRS、BmGlyRS和BmThrRS基因, 这些基因的功能可能由具有相似功能的其他蛋白完成, 或通过某个BmaaRS基因的可变剪接分别形成不同功能的BmaaRS。EST证据表明, BmaaRS基因存在不同形式的可变剪接; BmaaRS氨基酸序列的相似性及二、三级结构分析表明部分BmaaRS存在结构域的扩增, 有些不同的BmaaRS具有相同结构域, 相同功能的BmaaRS具有相似的三级结构; 进化分析表明, BmaaRS为2套不同来源的BmaaRS基因编码, 细胞质和线粒体BmaaRS的起源不同。     

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.     

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.     

tRNA-dependent active site assembly in a class I aminoacyl-tRNA synthetase

The crystal structure of ligand-free E. coli glutaminyl-tRNA synthetase (GlnRS) at 2.4 A resolution shows that substrate binding is essential to construction of a catalytically proficient active site. tRNA binding generates structural changes throughout the enzyme, repositioning key active site peptides that bind glutamine and ATP. The structure gives insight into longstanding questions regarding the tRNA dependence of glutaminyl adenylate formation, the coupling of amino acid and tRNA selectivities, and the roles of specific pathways for transmission of tRNA binding signals to the active site. Comparative analysis of the unliganded and tRNA-bound structures shows, in detail, how flexibility is built into the enzyme architecture and suggests that the induced-fit transitions are a key underlying determinant of both amino acid and tRNA specificity.     

Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase

Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino-acid activation and tRNA esterification are corrected by aminoacyl-tRNA synthetase-catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino-acid discrimination is less well understood. We show that phenylalanyl-tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl-adenylate and Tyr-tRNA(Phe). Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the beta subunit, 40 angstroms from the active site in the alpha subunit. Replacements of residues within the editing site had no effect on Phe-tRNA(Phe) synthesis, but abolished hydrolysis of Tyr-tRNA(Phe) in vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded beta-galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino-acid discrimination in vivo revealed that editing by phenylalanyl-tRNA synthetase is essential for faithful translation of the genetic code.     

tRNA sulfurtransferase: a member of the aminoacyl-tRNA synthetase complex in rat liver

Transfer RNA sulfurtransferase, tRNA methyltransferase, and aminoacyl-tRNA synthetase activity are associated in a complex in rat liver, which is excluded from Sephadex G-200 columns. The complex can also be isolated by subjecting cell supernatants to further centrifugation at 160,000 x g for 18 hours. The resulting pellet contains 70% of the total sulfurtransferase activity, and a 3-fold increase in specific activity is accomplished through pelleting. The data suggest that the enzymes of tRNA metabolism are organized in a large complex in rat liver.     

Occurrence of aminoacyl-tRNA synthetase complexes in calf brain

Eighteen aminoacyl-tRNA synthetases of the postribosomal supernatant fraction of brain cortex were characterized by glycerol density gradient centrifugation and gel filtration analysis. On the basis of sedimentation properties and gel elution profiles, four groups of enzyme activities were determined in the postribosomal supernatant fraction; the first group sedimenting at about 6 S contained 18 individual synthetase activities, the next successive groups of greater molecular sizes contained synthetase complexes, and the last group possessed activities of 15 synthetases. Each aminoacyl-tRNA synthetase appeared at least in two forms: free and bound in complexes of varying sizes and different enzyme compositions. Conventional purification methods of lysyl-tRNA synthetase from the post-ribosomal supernatant fraction of brain cortex gave a preparation containing four groups of aminoacylation activities. The obtained preparation contained a large complex, reduced number of intermediate complexes and some individual synthetases.     

Structure-functional properties of eukaryotic aminoacyl-tRNA synthetase]

Data on structural and functional peculiarities of eukaryotic aminoacyl-tRNA synthetases (structure, supermolecular organization, and localization in eukaryotic cell) are reviewed. The functional significance of aminoacyl-tRNA synthetase association with high molecular weight complexes and other cellular components is discussed.     

A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.     

Amino acid-dependent transfer RNA affinity in a class I aminoacyl-tRNA synthetase

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.     

Altered aminoacyl-tRNA synthetase complexes in CHO cell mutants

The Chinese hamster ovary (CHO) aminoacyl-tRNA synthetase mutants Gln-2, His-1, and Lys-101 were analyzed for alterations in respective particulate enzyme forms. The mutant Gln-2 showed a preferential loss of the lower molecular weight enzyme form for glutamine. His-1 showed alterations of the enzyme complexes for several other aminoacyl-tRNA activities but only decreased activity for itself. The mutant Lys-101 only showed an altered Lysyl-tRNA synthetase. These results provide evidence for a model of the intracellular role of the aminoacyl-tRNA synthetase complexes wherein the high molecular weight forms utilize amino acids directly from the extracellular pool while the low molecular weight forms utilize intracellular pools.     

An aminoacyl-tRNA synthetase complex in Escherichia coli.

Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M). In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran. The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers. The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons. Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis. Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl). Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases. These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation.