Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis.

With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique. Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis. The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E. coli threonyl-tRNA synthetase (42 and 47%). Both proteins complement an E. coli thrS mutant and effectively charge E. coli threonyl tRNA in vitro. Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B. subtilis chromosome. The regulatory regions of both genes are quite complex and show structural similarities. During vegetative growth, only the thrSv gene is expressed.     

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family.

We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.     

Unilateral aminoacylation specificity between bovine mitochondria and eubacteria

The present study shows unilateral aminoacylation specificity between bovine mitochondria and eubacteria (Escherichia coli and Thermus thermophilus) in five amino acid-specific aminoacylation systems. Mitochondrial synthetases were capable of charging eubacterial tRNA as well as mitochondrial tRNA, whereas eubacterial synthetases did not efficiently charge mitochondrial tRNA. Mitochondrial phenylalanyl-, threonyl-, arginyl-, and lysyl-tRNA synthetases were shown to charge and discriminate cognate E. coli tRNA species from noncognate ones strictly, as did the corresponding E. coli synthetases. By contrast, mitochondrial seryl-tRNA synthetase not only charged cognate E. coli serine tRNA species but also extensively misacylated noncognate E. coli tRNA species. These results suggest a certain conservation of tRNA recognition mechanisms between the mitochondrial and E. coli aminoacyl-tRNA synthetases in that anticodon sequences are most likely to be recognized by the former four synthetases, but not sufficiently by the seryl-tRNA synthetase. The unilaterality in aminoacylation may imply that tRNA recognition mechanisms of the mitochondrial synthetases have evolved to be, to some extent, simpler than their eubacterial counterparts in response to simplifications in the species-number and the structural elements of animal mitochondrial tRNAs.     

Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase

The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino-acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene.     

Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase

Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria.     

tRNA glycylation system from Thermus thermophilus. tRNAGly identity and functional interrelation with the glycylation systems from other phylae.

The systems of tRNA glycylation belong to the most complex aminoacylation systems since neither the oligomeric structure of glycyl-tRNA synthetases (GlyRS) nor the discriminator bases in tRNAGly are conserved in the phylae. To better understand the structure-function relationship in glycylation systems of various origins and the functional peculiarities related to their structural divergences, the elements in tRNA conferring its glycine identity in Thermus thermophilus were characterized and compared to those of other systems. Thermophilic identity is conferred by the G1-C72, C2-G71, G3-C70, and C50-G64 pairs together with the G10, U16, C35, and C36 single residues. In contrast to most other aminoacylation systems, the discriminator base is not directly involved in identity. Transplantation of these elements in tRNAAsp and tRNAPhe converts specificity toward glycine albeit conservation of nucleotide 73. Analysis of the functional interrelation of the identity elements shows coupling in synthetase recognition of the elements from anticodon and G10 whereas those from acceptor arm are recognized independently. Despite nondirect implication in identity, the discriminator base contributes cooperatively with C36 in specificity of glycylation. The link between the structural heterogeneity and the functional divergence of the glycylation systems and the phylogenic interrelation of these systems were approached by comparing the ability of GlyRSs of various phylae to glycylate heterologous tRNAGly. Dimeric GlyRSs from mammalian and archaebacteria acylate efficiently only eukaryotic and archaebacterial tRNAGly with a discriminatory A73, whereas tetrameric Escherichia coli GlyRS acylates only eubacterial tRNAGly with a discriminatory U73. In contrast, dimeric yeast GlyRS acylates efficiently both eukaryotic and archaebacterial tRNAGly as well as peculiar prokaryotic isoacceptors. Species specificity is lost with the dimeric GlyRS from Thermus thermophilus that acylates efficiently eubacterial, archaebacterial, and eukaryotic tRNAGly. These features are discussed in the context of the evolution of the glycylation systems and the phylogenic interrelation of the organisms.     

Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8

Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8. As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E. coli and B. stearothermophilus. Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T. thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not. The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another. The conformational transition involving the anticodon of E. coli tRNAGlu as complexed with Glu-tRNA synthetase from T. thermophilus is necessary for the aminoacylation activity.     

Autogenous repression of Escherichia coli threonyl-tRNA synthetase expression in vitro

Escherichia coli threonyl-tRNA synthetase (EC 6.1.1.3) expression has been examined in an acellular protein-synthesizing system programmed with a plasmid DNA carrying thrS, infC, pheS, and pheT, the gene for threonyl-tRNA synthetase, initiation factor 3, and the two protomers of phenylalanyl-tRNA synthetase (EC 6.1.1.20), respectively. The initial rate of synthesis of L-[35S]methionine-labeled threonyl-tRNA synthetase is markedly reduced by the addition of homogeneous RNase-free threonyl-tRNA synthetase to the assay, not by that of phenylanyl- or tyrosyl-tRNA synthetase (EC 6.1.1.1). The inhibition is 50% in the presence of 0.25 microM threonyl-tRNA synthetase and reaches 90% with 2 microM enzyme. Synthesis of mRNA in the acellular DNA-dependent protein-synthesizing system has been measured by molecular hybridization to gene-specific lambda DNA probes corresponding to thrS, pheS, and pheT. The addition to the assay of 2 microM threonyl-tRNA synthetase does not affect the extent of mRNA hybridizing to the thrS-specific DNA probe. This result is interpreted as reflecting an effect of the synthetase on its expression at the translational level. Analysis of the DNA sequence of the thrS gene predicts several potential secondary structures capable of forming in the thrS mRNA. One of these potential structures is a cloverleaf. The possible role of such structures in controlling expression of thrS is discussed.     

Interdomain compactization in human tyrosyl-tRNA synthetase studied by the hierarchical rotations technique

Aminoacyl-tRNA synthetases are key enzymes of protein biosynthesis which usually possess multidomain structures. Mammalian tyrosyl-tRNA synthetase is composed of two structural modules: N-terminal catalytic core and an EMAPII-like C-terminal domain separated by long flexible linker. The structure of full-length human cytoplasmic tyrosyl-tRNA synthetase is still unknown. The structures of isolated N-terminal and C-terminal domains of the protein are resolved, but their compact packing in a functional enzyme is a subject of debates. In this work we studied putative compactization of the N- and C-terminal modules of human tyrosyl-tRNA synthetase by the coarse-grained hierarchical rotations technique (HIEROT). The large number of distinct types of binding interfaces between N- and C-terminal modules is revealed in the absence of enzyme substrates. The binding propensities of different residues are computed and several binding "hot spots" are observed on the surfaces of N and C modules. These results could be used to govern atomistic molecular dynamics simulations, which will sample preferable binding interfaces effectively.     

Glutamyl-tRNA synthetase from Thermus thermophilus HB8. Molecular cloning of the gltX gene and crystallization of the overproduced protein.

The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.     

Effect of the overproduction of phenylalanyl- and threonyl-tRNA synthetases on tRNAPhe and tRNAThr concentrations in E. coli cells

Transformation of an E. coli strain with a recombinant plasmid DNA (pB1) encoding the genes for phenylalanyl- and threonyl-tRNA synthetases causes overproduction of these enzymes by about 100- and 5-fold, respectively. A possible effect of the overproduction of the two aminoacyl-tRNA synthetases on intracellular cognate tRNA levels has been searched for by comparing tRNAThr and tRNAPhe aminoacylation capacities in the RNA extracts from strains carrying pB1 or pBR322 plasmid DNA. The answer is that the levels of these tRNAs are not changed by selective increase of the cognate synthetases.     

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.     

Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases

Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain.     

Crystal structure of human mitochondrial tyrosyl-tRNA synthetase reveals common and idiosyncratic features

We report the structure of a strictly mitochondrial human synthetase, namely tyrosyl-tRNA synthetase (mt-TyrRS), in complex with an adenylate analog at 2.2 A resolution. The structure is that of an active enzyme deprived of the C-terminal S4-like domain and resembles eubacterial TyrRSs with a canonical tyrosine-binding pocket and adenylate-binding residues typical of class I synthetases. Two bulges at the enzyme surface, not seen in eubacterial TyrRSs, correspond to conserved sequences in mt-TyrRSs. The synthetase electrostatic surface potential differs from that of other TyrRSs, including the human cytoplasmic homolog and the mitochondrial one from Neurospora crassa. The homodimeric human mt-TyrRS shows an asymmetry propagating from the dimer interface toward the two catalytic sites and extremities of each subunit. Mutagenesis of the catalytic domain reveals functional importance of Ser200 in line with an involvement of A73 rather than N1-N72 in tyrosine identity.     

Consensus structure and evolution of 5S rRNA

A consensus structure model of 5S rRNA presenting all conserved nucleotides in fixed positions has been deduced from the primary and secondary structure of 71 eubacterial, archaebacterial, eukaryotic cytosolic and organellar molecules. Phylogenetically related groups of molecules are characterized by nucleotide deletions in helices III, IV and V, and by potential base pair interactions in helix IV. The group-specific deletions are correlated with the early branching pattern of a dendrogram calculated from nucleotide substitution data: the first major division separates the group of eubacterial and organellar molecules from a second group containing the common ancestors of archaebacterial and eukaryotic/cytosolic molecules. The earliest diverging branch of the eubacterial/organellar group includes molecules from Thermus thermophilus, T. aquaticus, Rhodospirillum rubrum, Paracoccus denitrificans and wheat mitochondria.     

Domain-domain communication for tRNA aminoacylation: the importance of covalent connectivity

Aminoacyl-tRNA synthetases form complexes with tRNA to catalyze transfer of activated amino acids to the 3' end of tRNA. The tRNA synthetase complexes are roughly divided into the activation and tRNA-binding domains of synthetases, which interact with the acceptor and anticodon ends of tRNAs, respectively. Efficient aminoacylation of tRNA by Escherichia coli cysteinyl-tRNA synthetase (CysRS) requires both domains, although the pathways for the long-range domain-domain communication are not well understood. Previous studies show that dissection of tRNA(Cys) into acceptor and anticodon helices seriously reduces the efficiency of aminoacylation, suggesting that communication requires covalent continuity of the tRNA backbone. Here we tested if communication requires the continuity of the synthetase backbone. Two N-terminal fragments and one C-terminal fragment of E. coli CysRS were generated. While the N-terminal fragments were active in adenylate synthesis, they were severely defective in the catalytic efficiency and specificity of tRNA aminoacylation. Conversely, although the C-terminal fragment was not catalytically active, it was able to bind and discriminate tRNA. However, addition of the C-terminal fragment to an N-terminal fragment in trans did not improve the aminoacylation efficiency of the N-terminal fragment to the level of the full-length enzyme. These results emphasize the importance of covalent continuity of both CysRS and tRNA(Cys) for efficient tRNA aminoacylation, and highlight the energetic costs of constraining the tRNA synthetase complex for domain-domain communication. Importantly, this study also provides new insights into the existence of several natural "split" synthetases that are now identified from genomic sequencing projects.     

Engineering and characterization of the ribosomal L10.L12 stalk complex. A structural element responsible for high turnover of the elongation factor G-dependent GTPase

The ribosomal stalk protein L12 is essential for events dependent on the GTP-binding translation factors. It has been recently shown that ribosomes from Thermus thermophilus contain a heptameric complex L10.(L12)2.(L12)2.(L12)2, rather than the conventional pentameric complex L10.(L12)2.(L12)2. Here we describe the reconstitution of the heptameric complex from purified L10 and L12 and the characterization of its role in elongation factor G-dependent GTPase activity using a hybrid system with Escherichia coli ribosomes. The T. thermophilus heptameric complex resulted in a 2.5-fold higher activity than the E. coli pentameric complex. The structural element of the T. thermophilus complex responsible for the higher activity was investigated using a chimeric L10 protein (Ec-Tt-L10), in which the C-terminal L12-binding site in E. coli L10 was replaced with the same region from T. thermophilus, and two chimeric L12 proteins: Ec-Tt-L12, in which the E. coli N-terminal domain was fused with the T. thermophilus C-terminal domain, and Tt.Ec-L12, in which the T. thermophilus N-terminal domain was fused with the E. coli C-terminal domain. High GTPase turnover was observed with the pentameric chimeric complex formed from E. coli L10 and Ec-Tt-L12 but not with the heptameric complex formed from Ec-Tt-L10 and Tt.Ec-L12. This suggested that the C-terminal region of T. thermophilus L12, rather than the heptameric nature of the complex, was responsible for the high GTPase turnover. Further analyses with other chimeric L12 proteins identified helix alpha6 as the region most likely to contain the responsible element.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.