The specificities of four yeast dihydrouridine synthases for cytoplasmic tRNAs

Dihydrouridine is a highly abundant modified nucleoside found widely in tRNAs of eubacteria, eukaryotes, and some archaea. In cytoplasmic tRNA of Saccharomyces cerevisiae, dihydrouridine occurs exclusively at positions 16, 17, 20, 20A, 20B, and 47. Here we show that the known dihydrouridine synthases Dus1p and Dus2p and two previously uncharacterized homologs, Dus3p (encoded by YLR401c) and Dus4p (YLR405w), are required for all of the dihydrouridine modification of cytoplasmic tRNAs in S. cerevisiae. We have mapped the in vivo position specificity of the four Dus proteins, by three complementary approaches: determination of the molar ratio of dihydrouridine in purified tRNAs from different dus mutants; microarray analysis of a large number of tRNAs based on differential hybridization of uridine and dihydrouridine-containing tRNAs to the complementary oligonucleotides; and the development and use of a novel dihydrouridine mapping technique, employing primer extension. We show that each of the four Dus proteins has a distinct position specificity: Dus1p for U(16) and U(17), Dus2p for U(20), Dus3p for U(47), and Dus4p for U(20a) and U(20b).     

Recognition of tRNAs by Methionyl-tRNA transformylase from mammalian mitochondria

Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator. In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNA(Met). In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA(f)(Met) mainly involves a set of nucleotides in the acceptor stem, particularly a C(1)A(72) mismatch. In animal mitochondria, only one tRNA(Met) species has yet been described. It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group. In the present study, we searched for the identity elements of tRNA(Met) that govern its formylation by bovine mitochondrial transformylase. The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate. Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNA(Met) an A(1)U(72) motif.     

Mechanism of functioning of aminoacyl-tRNA-synthetases

The article reviews the data available concerning the kinetics of reactions catalysed by eukaryotic and procaryotic aminoacyl-tRNA synthetases (E.C.6.1.1). It is shown that most of these enzymes operate in accordance with the flip-flop mechanism although individual variations do exist. It is stressed that a simplified kinetic approach of Cleland is inappropriate if applied to the aminoacyl-tRNA synthetases where subunit interactions are involved in the overall kinetics. The fidelity of aminoacylation reaction depends not only on the proofreading mechanisms at catalytic centres but also on the contribution of mutual conformational changes induced in interacting subunits of the synthetases.     

Interaction of aminoacyl-tRNA-synthetases with amino acids

The review deals with interactions of the key enzymes of the protein biosynthesis-aminoacyl-tRNA synthetases (EC 6.1.1.) with amino acids and their analogues, considering the contribution of different groups in the process of specific complex formation and catalysis. The important role of alpha-amino group of amino acid in the enzyme recognition has been revealed. Modification of the carboxylic group does not change significantly the analogues complex formation with aminoacyl-tRNA synthetases. However this group is essential for amino acid rearrangement in the specific complex with the enzyme. The structural organization of the enzyme binding sites specific for amino acids and the enzyme interaction with the analogues of aminoacyladenylates are discussed.     

Covalent derivatives of aminoacyl-tRNA-synthetases with substrates

The possibility of participation of covalent enzyme substrate's derivatives in reactions catalyzed by aminoacyl-tRNA synthetases is discussed in connection with general principles of enzyme catalysis. Tryptophanylated and pyrophosphorylated forms of beef pancreas tryptophanyl-tRNA synthetase were identified, isolated and characterized and the expected role of these derivatives in the enzyme function is discussed.     

Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities.

The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.     

Correlation between Two Glycine tRNAs and Corresponding Glycyl tRNA Synthetases

A gene encoding a stereo-specific secondary alcohol dehydrogenase (CpSADH) that catalyzed the oxidation of (S)-1,3-BDO to 4-hydroxy-2-butanone was cloned from Candida parapsilosis. This CpSADH-gene consisted of 1,009 nucleotides coding for a protein with M _r 35,964. A recombinant Escherichia coli JM109 strain harboring the expression plasmid, pKK-CPA1, produced (R)-1,3-BDO (93.5% ee, 94.7% yield) from the racemate without any additive to regenerate NAD⁺ from NADH.     

Exploring the specificity of bacterial elongation factor Tu for different tRNAs

In order to identify amino acids in Thermus thermophilus elongation factor Tu which contribute to its specificity for different tRNAs, the binding affinities of 20 point mutations were compared to that of wild type protein using four tRNAs of differing affinities. The observed specificity for tRNA is the result of the varying contributions of five amino acids which make contacts with the T-stem and the 3' terminus of tRNA. For three of the amino acids the test tRNAs differ in sequence at the site of contact, presumably explaining the specificity. However, the remaining two amino acids contact tRNA at conserved positions, suggesting that more global structural or dynamic properties of the free tRNA contribute to specificity.     

Recognition of tRNAs by aminoacyl-tRNA synthetases: Escherichia coli tRNAMet and E. coli methionyl-tRNA synthetase

In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. We have now investigated the aminoacylation activity of a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, which indicates that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.     

The accuracy of poly(U) translation by different eukaryotic tRNAs

In this work we present comparative data on rates of phenylalanine and leucine incorporation into the poly(U) dependent product of cell-free translation by different eukaryotic tRNAs at high Mg2+ concentration. The frequency of translation errors has been found to depend upon the value of the tRNAPhe:tRNALeu ratio and the peculiarities of isoacceptor tRNAsLeu of different origin.     

Two homologous fungal carbonyl reductases with different substrate specificities

Two homologous fungal short-chain dehydrogenase/reductase (SDR) proteins have been cloned from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus) and expressed in Escherichia coli: trihydroxynaphthalene reductase (3HNR), an enzyme of the melanin biosynthetic pathway that catalyzes the conversion of 1,3,8-trihydroxynaphthalene to vermelone, and 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl), which acts on androgens and estrogens, although its physiological substrate remains to be defined. In the present study, we have compared the structures, specificities to substrates and inhibitors, temperature and pH optima of 3HNR and 17beta-HSDcl. Sequence analysis and homology-built models revealed that these enzymes are highly similar. Both of these enzymes are NADP(H)-preferring reductases and act on steroids at position 17; however, 17beta-HSDcl presented considerably higher initial rates than 3HNR. In vitro, 17beta-HSDcl preferably catalyzed the reduction of 4-estrene-3,17-dione, while the best steroid substrate for 3HNR was 5alpha-androstane-3,17-dione. On the other hand, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), an artificial substrate of 3HNR, was oxidized rapidly by 3HNR, while it was not a substrate for 17beta-HSDcl. Additionally, our data show that tricyclazole, a specific inhibitor of 3HNR, is 100-fold less effective for 17beta-HSDcl inhibition, while flavonoids can inhibit both 3HNR and 17beta-HSDcl. We have also examined the effects of temperature and pH on the oxidation of DDBO by 3HNR and the oxidation of 4-estrene-17beta-ol-3-one by 17beta-HSDcl. The apparent optimal temperature for 3HNR activity was between 25 and 30 degrees C, while it was between 40 and 45 degrees C for 17beta-HSDcl activity. The pH optimum of 3HNR activity was between 8 and 9, and for 17beta-HSDcl, between 7 and 8. Our data show that in spite of high homology and similar backbone structure, differences between 3HNR and 17beta-HSDcl were not only in substrate specificities, but also in temperature and pH optima.