The binding of aminoacyl-tRNA synthetases to triazine dye conjugates.

The binding of thirteen aminoacyl-tRNA synthetases to thirty two immobilised procion dyes has been investigated. Most dyes bind one or more enzymes. The amino acid substrates are not normally potent eluants, with the notable exception of tryptophan eluting tryptophanyl-tRNA synthetase from Brown MX-5BR. Phosphate is frequently extremely effective, much more than expected by simple considerations of ionic strength, indicating that many of the dyes are able to mimic the phosphate groups of the phosphodiester backbone of the nucleic acid. Procedures for the purification of methionyl-, tryptophanyl- and tyrosyl-tRNA synthetases are presented and compared to the conventional purifications of these enzymes. The results indicate the general applicability of these dye columns to the purification of most enzymes of of nucleic acid metabolism and the necessity of investigating as many different dyes as possible for any individual enzyme.     

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.     

The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.

The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.     

Synthesis of cysteine-containing dipeptides by aminoacyl-tRNA synthetases.

Arginyl-tRNA synthetase (ArgRS) catalyses AMP- and PPi-independent deacylation of Arg-tRNAArg in the presence of cysteine. A dipeptide, Arg-Cys, is a product of this deacylation reaction. Similar reaction with homocysteine yields Arg-Hcy. Arginine is a noncompetitive inhibitor of the cysteine-dependent deacylation which indicates that cysteine binds to the enzyme-Arg-tRNAArg complex at a site separate from the arginine binding site. In the presence of arginine, [14C]Arg-tRNAArg is deacylated at a rate similar to the rate of its spontaneous deacylation in solution and [14C]arginine is a product. Experiments with cysteine derivatives indicate that the -SH group is essential for the reaction whereas -NH2 and -COOH groups are not. Thioesters of arginine are formed with 3-mercaptopropionic acid, N-acetyl-L-cysteine and dithiothreitol. These data suggest that formation of the dipeptide Arg-Cys involves a thioester intermediate, S-(L-arginyl)-L-cysteine, which is not observed because of the rapid rearrangement to form a stable peptide bond. Facile intramolecular reaction results from the favorable geometric arrangement of the alpha-amino group of cysteine with respect to the thioester formed in the initial reaction. Similar reactions, yielding Ile-Cys and Val-Cys, are catalyzed by isoleucyl- and valyl-tRNA synthetases, respectively.     

Immunochemical quantification of procion red HE-3B used as ligand in affinity chromatography.

The quantification of Procion Red HE-3B used as a ligand in affinity chromatography for proteins is reported. It's based on an enzyme-linked immunosorbent assay using antibodies against the dye. Polyclonal antibodies were classically prepared after conjugation of the dye on KLH and injection into rabbits. The development of the assay was based on the competitive inhibition between hemoglobin-dye complex and free dye. The sensitivity of this method was about 1000-times higher than a classical spectrophotometric assay, and was modulated by some chemical substituents attached on the native dye. It was demonstrated that the assay was applicable to the determination of dye traces that may be released from dye affinity sorbents. Moreover, the quantification of the dye was successfully applied to proteins that are being purified from a dye affinity column.     

Nuclear origin of specific yeast mitochondrial aminoacyl-tRNA synthetases.

Hydroxylapatite chromatographies of mitochondrial and total enzymes from a rho+ yeast, or from the related rho degrees mitochondrial DNA-less mutant, show the occurrence in the mitochondrial enzyme of one Phe-, one Met-, one Leu-tRNA synthetase peak which elutes distinctly from the cytoplasmic counterpart and charges well mitochondrial tRNA, whereas the cytoplasmic enzyme does not. The measurement of the mitochondrial synthetases activities in various enzymatic extracts shows that they are not repressed in rho+ cells grown on 10% glucose and that they are concentrated in the mitochondria (Phe- and Met- tRNA synthetases) but are also present outside the mitochondria. It is concluded that yeast mitochondrial protein biosynthesis involves the nuclear coded mitochondrial specific Phe-, Met- and Leu-tRNA synthetases and that the entrance of the synthetases into the mitochondria needs no factor depending on the mitochondrial DNA.     

Fractionation of plant aminoacyl-tRNA synthetases on tRNA-Sepharose columns.

It has been shown that tRNA-Sepharose, a chromatographic adsorbent containing unfractionated tRNA bound to a Sepharose matrix, is a useful, group-specific adsorbent for fractionation of the plant aminoacyl-tRNA synthetases. Conditions are described in which Val-, Trp-, Phe-, Leu- and Ile-tRNA synthetases from yellow lupin seeds can be separated from each other on the tRNA-Sepharose columns. Factors affecting affinity chromatography on the t-RNA-Sepharose columns are discussed. The affinity chromatography procedure for the purification of lupin Ser-tRNA synthetase to homogenity is described.     

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.     

Sequence similarities among the family of aminoacyl-tRNA synthetases

Recent affinity labeling studies have led to the identification of lysine residues at the CCA binding site of tRNA in Escherichia coli methionyl- and tyrosyl-tRNA synthetases. The comparison of the labeled peptides to the known primary structures of the aminoacyl-tRNA synthetases reveals new sequence similarities among this family of enzymes. These similarities include a 'constant' lysine residue whose functional significance is discussed. Moreover, a systematic computer analysis was conducted to search for similarities between the aminoacyl-tRNA synthetases taken as pairs.     

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.     

Origin and evolution of the mitochondrial aminoacyl-tRNA synthetases

Many theories favor a fusion of 2 prokaryotic genomes for the origin of the Eukaryotes, but there are disagreements on the origin, timing, and cellular structures of the cells involved. Equally controversial is the source of the nuclear genes for mitochondrial proteins, although the alpha-proteobacterial contribution to the mitochondrial genome is well established. Phylogenetic inferences show that the nuclearly encoded mitochondrial aminoacyl-tRNA synthetases (aaRSs) occupy a position in the tree that is not close to any of the currently sequenced alpha-proteobacterial genomes, despite cohesive and remarkably well-resolved alpha-proteobacterial clades in 12 of the 20 trees. Two or more alpha-proteobacterial clusters were observed in 8 cases, indicative of differential loss of paralogous genes or horizontal gene transfer. Replacement and retargeting events within the nuclear genomes of the Eukaryotes was indicated in 10 trees, 4 of which also show split alpha-proteobacterial groups. A majority of the mitochondrial aaRSs originate from within the bacterial domain, but none specifically from the alpha-Proteobacteria. For some aaRS, the endosymbiotic origin may have been erased by ongoing gene replacements on the bacterial as well as the eukaryotic side. For others that accurately resolve the alpha-proteobacterial divergence patterns, the lack of affiliation with mitochondria is more surprising. We hypothesize that the ancestral eukaryotic gene pool hosted primordial "bacterial-like" genes, to which a limited set of alpha-proteobacterial genes, mostly coding for components of the respiratory chain complexes, were added and selectively maintained.     

Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin

The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.     

Mechanisms of molecular recognition of tRNAs by aminoacyl-tRNA synthetases.

Interactions of Escherichia coli isoleucyl- and glutamyl-tRNA synthetases and their cognate tRNAs were analyzed by phosphate-alkylation mapping with N-nitroso-N-ethylurea and/or by 1H-NMR analysis. When E. coli tRNA(Ile) was bound with isoleucyl-tRNA synthetase, many of the phosphate groups in the anticodon loop and stem and in the D-stem were protected from alkylation. This result is consistent with that of analysis of imino proton resonances due to the secondary and tertiary base pairs. These analyses also suggested that the L-shaped tertiary structure of tRNA(Ile) is distorted upon complex formation with IleRS because of disruption of some tertiary base pairs. In the case of E. coli tRNA(Glu), several phosphate groups in the D-stem and the variable loop were significantly protected by the cognate synthetase. These results indicate that the two tRNAs, unlike other tRNAs studied so far, have some of the "identity determinants" in the D-stem and/or in the anticodon stem.     

Partial reactions of aminoacyl-tRNA synthetases as functions of pH.

The effect of pH on the properties of the partial reactions of arginyl-tRNA synthetase of E. coli has been investigated. V max of pyrophosphorolysis of arginyl adenylate has a pH optimum at pH 6.1, whereas V max of the transfer of arginine to tRNA has a pH optimum of 8.2. These values correlate with the pH optima of the ATP:PPi exchange and the overall esterification reaction, respectively. Only the pyrophosphorolysis reaction requires a divalent cation; transfer proceeds in the presence of EDTA. Inorganic pyrophosphate inhibits the transfer reaction to an extent independent of the concentration of tRNA; the maximum inhibition is a function of pH, corresponding to the relative rate of pyrophosphorolysis of the common intermediate compared with the rate of transfer. These results show that different groups on the enzyme participate in the rate-limiting steps of the two partial reactions and that these partial reactions have properties consistent with their participation in the overall esterification of arginine with tRNA.     

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation.

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

Relationship of the CCA sequence of tRNA with the early evolutional aspect of aminoacyl-tRNA synthetases.

The CCA sequence is common to the 3'-ends of all tRNAs. We investigated the requirement of the CCA sequence in aminoacylation with the cognate aminoacyl-tRNA synthetases (aaRSs) and several interesting conclusions could be drawn. In tRNAs belonging to the class I aaRSs, decreased aminoacylation activities resulted from the substitution of A76 with a pyrimidine, whereas in tRNAs belonging to the class II aaRSs, decreased aminoacylation activities resulted from the substitution with guanine. The results suggest that aminoacylation of proto-tRNA might have started through the direct hydrophobic (or stacking) interaction between the large, hydrophobic amino acid residue (now utilizing a class I aaRS) of aminoacyl-AMP and the 3'-terminal adenine. The shorter distance between the adenine and the 2'-OH position than the 3'-OH position, and the bulkiness and hydrophobicity of amino acids may be important reasons why class I aaRSs select the 2'-OH position in aminoacylation. Molecular mechanics-based conformation modeling also indicated that the resulting positioning of the adenine and the amino acid residue of 2'-aminoacyl-adenosine for large amino acid is in the vicinity. In contrast, in the case of small amino acids (with class II aaRSs) which would not be able to use the hydrophobic interaction, a protein enzyme might have participated in the aminoacylation reaction from an early stage. The active-site folds of aaRSs belonging to each class reflect the history of evolution: typical nucleotide-binding fold (Rossman fold) in the case of class I aaRSs, and primitive fold which is found also among the family of nonribosomal peptide synthetases in the case of class II aaRSs.