Association of eukaryotic aminoacyl-tRNA-synthases with polyribosomes

Upon fractionation of a mitochondria-free extract of rabbit reticulocytes into a ribosome-free extract and mono- and polyribosomes the bulk of the aminoacyl-tRNA synthetase activity was found in the fraction of mono- and polyribosomes. All the fifteen aminoacyl-tRNA synthetases were revealed, although in somewhat different quantities, in both fractions of the mitochondria-free reticulocyte extract. Aminoacyl-tRNA synthetases of the ribosome-free extract are found in two forms: RNA-binding one, and, the one having no affinity for high molecular weight RNAs. Aminoacyl-tRNA synthetases dissociated from the complexes with polyribosomes exist only in the RNA-binding form. All aminoacyl-tRNA synthetases can be removed from such complexes by an addition of 16S rRNA of E. coli, poly(U) or tRNA of rabbit reticulocytes. This testifies to labile association of aminoacyl-tRNA synthetases with the RNA-component of polyribosomes as well as to a rather nonspecific character of their interaction. After EDTA-induced dissociation of polyribosomes, the aminoacyl-tRNA synthetase activity was detected in the complex with both ribosomal subunits.     

Structure of Hsp15 reveals a novel RNA-binding motif

We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome. Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif. This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA. The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site. An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif. This may imply a hitherto unrecognized functional similarity between these three protein classes.     

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.     

Crystal structure of Mil (Mth680): internal duplication and similarity between the Imp4/Brix domain and the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases

Proteins of the Imp4/Brix superfamily are involved in ribosomal RNA processing, an essential function in all cells. We report the first structure of an Imp4/Brix superfamily protein, the Mil (for Methanothermobacter thermautotrophicus Imp4-like) protein (gene product Mth680), from the archaeon M. thermautotrophicus. The amino- and carboxy-terminal halves of Mil show significant structural similarity to one another, suggesting an origin by means of an ancestral duplication. Both halves show the same fold as the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases, with greater conservation seen in the N-terminal half. This structural similarity, together with the charge distribution in Mil, suggests that Imp4/Brix superfamily proteins could bind single-stranded segments of RNA along a concave surface formed by the N-terminal half of their beta-sheet and a central alpha-helix. The crystal structure of Mil is incompatible with the presence, in the Imp4/Brix domain, of a helix-turn-helix motif that was proposed to comprise the RNA-binding moiety of the Imp4/Brix proteins.     

Development of structural organization of protein-synthesizing machinery from prokaryotes to eukaryotes

Though the mechanisms of protein biosynthesis are similar in the cells of prokaryotes and eukaryotes, the eukaryotic translational machinery in the cell is arranged more intricately. One of the most striking characteristic features of the eukaryotic translational machinery is that the eukaryotic proteins involved in the translational process, such as initiation factors, elongation factors and aminoacyl-tRNA synthetases, in contrast to their prokaryotic analogs, possess a non-specific affinity for RNA. Due to the RNA-binding ability, these eukaryotic proteins can be compartmentalized on polyribosomes. In addition to the proteins of the translational apparatus, several other eukaryotic RNA-binding proteins can be also compartmentalized on polyribosomes; these proteins include glycolytic enzymes, steroid hormone receptors and intermediate filament proteins. Thus, the eukaryotic polyribosome is an element of the cytoplasmic labile structure on which various proteins can be compartmentalized and, consequently, different biochemical pathways can be integrated.     

Duplication and Quadruplication of Arabidopsis thaliana Cysteinyl- and Asparaginyl-tRNA Synthetase Genes of Organellar Origin

Two cysteinyl-tRNA synthetases (CysRS) and four asparaginyl-tRNA synthetases (AsnRS) from Arabidopsis thaliana were characterized from genome sequence data, EST sequences, and RACE sequences. For one CysRS and one AsnRS, sequence alignments and prediction programs suggested the presence of an N-terminal organellar targeting peptide. Transient expression of these putative targeting sequences joined to jellyfish green fluorescent protein (GFP) demonstrated that both presequences can efficiently dual-target GFP to mitochondria and plastids. The other CysRS and AsnRSs lack targeting sequences and presumably aminoacylate cytosolic tRNAs. Phylogenetic analysis suggests that the four AsnRSs evolved by repeated duplication of a gene transferred from an ancestral plastid and that the CysRSs also arose by duplication of a transferred organelle gene (possibly mitochondrial). These case histories are the best examples to date of capture of organellar aminoacyl-tRNA synthetases by the cytosolic protein synthesis machinery. Received: 8 October 1999 / Accepted: 23 January 2000     

Functional dissection of the eukaryotic-specific tRNA-interacting factor of lysyl-tRNA synthetase

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, referred to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNA-binding site. Their mutation into alanine led to a reduced affinity for tRNA(3)(Lys) or minimalized tRNA mimicking the acceptor-TPsiC stem-loop of tRNA(3)(Lys) and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA(Lys) networking during translation or to convey tRNA(3)(Lys) into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNA-interacting factor.     

Large-scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl-tRNA synthetase

Structural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a “closed” form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl-tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.     

Aminoacyl-tRNA synthetase-catalyzed cleavage of the glycosidic bond of 5-halogenated uridines.

Because of previous data suggesting that aminoacyl-tRNA synthetases make a transient Michael adduct with a specific uridine residue in the tRNA structure, (Schoemaker, H.J.P., and Schimmel, P.R. (1977) Biochemistry 16, 5454-5460) attempts were made to find simple model systems in which this reaction might be studied in more detail. In the course of these investigations, it was found that Escherichia coli Ile-tRNA synthetase catalyzes cleavage of the glycosidic bond of 5-bromouridine. At pH 7.5, ambient temperatures, the turnover number is roughly 5/h. 5-Fluoro-, 5-chloro-, and 5-iodouridine are also cleaved in an analogous way by Ile-tRNA synthetase. In the case of uridine, conversion of uridine to uracil and ribose was also detected, but with a smaller turnover number. Three other E. coli and one mammalian aminoacyl-tRNA synthetases were also examined and all were found to catalyze glycosidic bond cleavage of 5-bromouridine. The data indicate that, in general, synthetases have a catalytic center that shows an unusual reactivity for uridine.     

Multidomain enzymes involved in peptide synthesis.

Biosynthesis of peptides in non-ribosomal systems is catalyzed by multifunctional enzymes that employ the thio-template mechanism. Recent studies on the analysis of the primary structure of several peptide synthetases have revealed that they are organized in highly conserved and repeated functional domains. The aligned domains provide the template for peptide synthesis, and their order determines the sequence of the peptide product.     

A gene fusion event in the evolution of aminoacyl-tRNA synthetases

The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life. In bacteria and archaea, distinct genes encode the two proteins. In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein. The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases. Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl-tRNA synthetases. In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively. Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units.     

Functional characterization of naturally occurring genetic variations of the human guanine-rich RNA sequence binding factor 1 (GRSF1)

The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D–models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.     

On the evolution of structure in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Sequence analysis and modular organization of threonyl-tRNA synthetase from Thermus thermophilus and its interrelation with threonyl-tRNA synthetases of other origins.

The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr-tRNA synthetases. Comparative analysis with the three-dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core and a C-terminal anticodon-binding module. Comparison with the three-dimensional structure of Escherichia coli Thr-tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity. Despite strong similarites in modular organization, Thr-tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr-tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon-like stem-loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr-tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.     

On the Evolution of Structure in Aminoacyl-tRNA Synthetases

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Basic faced alpha-helices are widespread in the peptide extensions of the eukaryotic aminoacyl-tRNA synthetases

Three aminoacyl-tRNA synthetases from yeast, one from plants and one from mammals possess unusual structures at their N termini, namely alpha helices with basic residues distributed asymmetrically, on a single face of the helix. It is unknown if these 'basic faced' alpha helices (BFAHs) are unique to the aminoacyl-tRNA synthetases. Analysis of the amino acid sequences of these five aminoacyl-tRNA synthetases using the hydrophobic moment algorithm failed to accurately identify the BFAHs. A new algorithm was therefore developed, called the 'basic moment'. This is a Fourier analysis procedure that predicts the distribution of basic residues within protein secondary structure. The basic moment identifies with a high degree of accuracy the five known BFAHs and also identifies further potential BFAHs at evolutionarily conserved positions in the peptide extensions of aspartyl-, lysyl- and valyl- tRNA synthetases from a range of eukaryotic species. In addition, the algorithm identifies the two-helix pair tRNA binding domain of alanyl-tRNA synthetase, implying that the domain includes a BFAH. The functional and evolutionary aspects of these structural features are discussed.     

Free fatty acids associated with the high molecular weight aminoacyl-tRNA synthetase complex influence its structure and function

Aminoacyl-tRNA synthetases from higher eukaryotes often are isolated as high molecular weight complexes associated with other components such as lipids. Since hydrophobic interactions are involved in the organization of the complex, it has been suggested that interaction of synthetases with these lipids might be important for their structure and function. Delipidation is known to affect certain properties of synthetases within the complex including sensitivity to detergents plus salts, temperature inactivation, hydrophobicity, sensitivity to proteases, and, as shown here, sensitivity to p-mercuribenzoate and sites of papain cleavage. Of the lipids known to co-purify with the complex, cholesterol esters, phospholipids and free fatty acids, we show that the particular lipids responsible for many of these changes are the free fatty acids. Specific removal of fatty acids results in a complex with properties similar to one totally delipidated by detergent treatment, and readdition of the fatty acid fraction reverses the effects. The fatty acid fraction contains both saturated and unsaturated fatty acids, but unsaturated fatty acids are much more effective in reversing the properties of the delipidated complex that are saturated fatty acids. These results indicate that the free fatty acids co-purifying with the synthetase complex bind to the synthetases and affect their structure and function.     

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.     

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.