A single base pair affects binding and catalytic parameters in the molecular recognition of a transfer RNA

A single G3.U70 base pair in the acceptor helix is a major determinant of the identity of an alanine transfer RNA. Alteration of this base pair to A.U or G.C prevents aminoacylation with alanine. We show here that, at approximate physiological conditions (pH 7.5, 37 degrees C), high concentrations of the mutant A3.U70 species do not inhibit aminoacylation of a wild-type alanine tRNA. The observation suggests that, under these conditions, the G3 to A3 substitution increases Km for tRNA by more than 30-fold. Other experiments at pH 7.5 show that no aminoacylation of A3.U70, G3.C70, or U3.G70 mutant tRNAs occurs with substrate levels of enzyme. This suggests that kcat for these mutant tRNAs is sharply reduced as well and that the catalytic defect is not due to slow release of charged mutant tRNAs from the enzyme. Investigations were also done at pH 5.5, where association of tRNAs with synthetases is generally stronger and where binding can be conveniently measured apart from aminoacylation. Under these conditions, the binding of the A3.U70 and G3.C70 species is readily detected and is only 3-5-fold weaker than the binding of the wild-type tRNA. Although the A3.U70 species was demonstrated to compete with the wild-type tRNA for the same site on the enzyme, no aminoacylation could be detected. Thus, even when conditions are adjusted to obtain strong competitive binding, a sharp reduction in kcat prevents aminoacylation of a tRNA(Ala) species with a substitution at position 3.70.     

Maintaining genetic code through adaptations of tRNA synthetases to taxonomic domains

The universal genetic code is determined by the aminoacylation of tRNAs. In spite of the universality of the code, there are barriers to aminoacylation across taxonomic domains. These barriers are thought to correlate with the co-segregation of sequences of synthetases and tRNAs into distinct taxonomic domains. By contrast, we show here examples of eukaryote-like synthetases that are found in certain prokaryotes. The associated tRNAs have retained their prokaryote-like character in each instance. Thus, co-segregation of domain-specific synthetases and tRNAs does not always occur. Instead, synthetases make adaptations of tRNA-protein contacts to cross taxonomic domains.     

The many routes of bacterial transfer RNAs after aminoacylation

Subsequent to their aminoacylation, tRNAs are subject to specific maturation and/or correction processes. Aminoacylated tRNAs ready for use in translation are then specifically channelled to the ribosomal A or P sites. Structural and biochemical studies have opened the way towards furthering our understanding of these routes to the ribosome, which involve a strict distinction between initiator and elongator tRNAs.     

Study of the role of the acceptor stem in the interactions between tRNAs and aminoacyl-tRNA synthetases.

Several studies have clearly demonstrated that the end of the acceptor stem was a very important area determining the aminoacylation properties of tRNAs. However the attempts to measure the contribution of this region to the binding of tRNAs to aminoacyl-tRNA synthetases have led to contradictory results. We report here the stepwise degradation of yeast tRNA-Phe and tRNA-Val from their 3' terminus, up to the seventh nucleotide : the affinity of each of the degraded-tRNA for their cognate aminoacyl-tRNA synthetase was compared to that of intact tRNA and it was found that these affinities are not significantly decreased when compared to those of the intact tRNAs.     

Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.

We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E. coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change. Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E. coli GlnRS in vitro and in vivo. The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln). GlnRS complex. Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro. We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo. We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.     

Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems

The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.     

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.     

Fragile T-stem in disease-associated human mitochondrial tRNA sensitizes structure to local and distant mutations

Mutations in human mitochondrial isoleucine tRNA (hs mt tRNA(Ile)) are associated with cardiomyopathy and opthalmoplegia. A recent study showed that opthalmoplegia-related mutations gave rise to severe decreases in aminoacylation efficiencies and that the defective mutant tRNAs were effective inhibitors of aminoacylation of the wild-type substrate. The results suggested that the effectiveness of the mutations was due in large part to an inherently fragile mitochondrial tRNA structure. Here, we investigate mutant tRNAs associated with cardiomyopathy, and a series of rationally designed second-site substitutions introduced into both opthalmoplegia- and cardiomyopathy-related mutant tRNAs. A source of structural fragility was uncovered. An inherently unstable T-stem appears susceptible to misalignments. This susceptibility sensitizes both domains of the L-shaped tRNA structure to base substitutions that are deleterious. Thus, the fragile T-stem makes the structure of this human mitochondrial tRNA particularly vulnerable to local and distant mutations.     

Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. To prevent errors in protein synthesis, many synthetases have evolved editing pathways by which misactivated amino acids (pre-transfer editing) and misacylated tRNAs (post-transfer editing) are hydrolyzed. Previous studies have shown that class II prolyl-tRNA synthetase (ProRS) possesses both pre- and post-transfer editing functions against noncognate alanine. To assess the relative contributions of pre- and post-transfer editing, presented herein are kinetic studies of an Escherichia coli ProRS mutant in which post-transfer editing is selectively inactivated, effectively isolating the pre-transfer editing pathway. When post-transfer editing is abolished, substantial levels of alanine mischarging are observed under saturating amino acid conditions, indicating that pre-transfer editing alone cannot prevent the formation of Ala-tRNA Pro. Steady-state kinetic parameters for aminoacylation measured under these conditions reveal that the preference for proline over alanine is 2000-fold, which is well within the regime where editing is required. Simultaneous measurement of AMP and Ala-tRNA Pro formation in the presence of tRNA Pro suggested that misactivated alanine is efficiently transferred to tRNA to form the mischarged product. In the absence of tRNA, enzyme-catalyzed Ala-AMP hydrolysis is the dominant form of editing, with "selective release" of noncognate adenylate from the active site constituting a minor pathway. Studies with human and Methanococcus jannaschii ProRS, which lack a post-transfer editing domain, suggest that enzymatic pre-transfer editing occurs within the aminoacylation active site. Taken together, the results reported herein illustrate how both pre- and post-transfer editing pathways work in concert to ensure accurate aminoacylation by ProRS.     

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.     

Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.

For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.     

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.     

RNA minihelices and the decoding of genetic information

The rules of the genetic code are determined by the specific aminoacylation of transfer RNAs by aminoacyl transfer RNA synthetase. A straightforward analysis shows that a system of synthetase-tRNA interactions that relies on anticodons for specificity could, in principle, enable most synthetases to distinguish their cognate tRNA isoacceptors from all others. Although the anticodons of some tRNAs are recognition sites for the cognate aminoacyl tRNA synthetases, for other synthetases the anticodon is dispensable for specific aminoacylation. In particular, alanine and histidine tRNA synthetases aminoacylate small RNA minihelices that reconstruct the part of their cognate tRNAs that is proximate to the amino acid attachment site. Helices with as few as six base pairs can be efficiently aminoacylated. The specificity of aminoacylation is determined by a few nucleotides and can be converted from one amino acid to another by the change of only a few nucleotides. These findings suggest that, for a subgroup of the synthetases, there is a distinct code in the acceptor helix of transfer RNAs that determines aminoacylation specificity.     

Modeling with in vitro kinetic parameters for the elaboration of transfer RNA identity in vivo

A tRNA with "double identity" was created, and this tRNA was demonstrated in vitro to aminoacylate quantitatively with either of two amino acids. In contrast, acceptance of only one of these amino acids was observed in vivo, and a simple manipulation determined which one was accepted. Kinetic parameters were obtained for aminoacylation with each amino acid of the tRNA with double identity and of related tRNAs. Modeling with these parameters largely explains which amino acid specificity is observed in vivo. The results delineate some of the kinetic boundaries for the design and accommodation of tRNA sequence variations in the elaboration of identity in vivo.