Conservation of a tRNA core for aminoacylation

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.     

Ribozyme-catalyzed tRNA aminoacylation

The RNA world hypothesis implies that coded protein synthesis evolved from a set of ribozyme catalyzed acyl-transfer reactions, including those of aminoacyl-tRNA synthetase ribozymes. We report here that a bifunctional ribozyme generated by directed in vitro evolution can specifically recognize an activated glutaminyl ester and aminoacylate a targeted tRNA, via a covalent aminoacyl-ribozyme intermediate. The ribozyme consists of two distinct catalytic domains; one domain recognizes the glutamine substrate and self-aminoacylates its own 5'-hydroxyl group, and the other recognizes the tRNA and transfers the aminoacyl group to the 3'-end. The interaction of these domains results in a unique pseudoknotted structure, and the ribozyme requires a change in conformation to perform the sequential aminoacylation reactions. Our result supports the idea that aminoacyl-tRNA synthetase ribozymes could have played a key role in the evolution of the genetic code and RNA-directed translation.     

Indirect readout of tRNA for aminoacylation

Aminoacylation of tRNA by aminoacyl-tRNA synthetases is the essential reaction that matches protein amino acids with the trinucleotide sequences specified in mRNA. Direct electrostatic interactions made by tRNA synthetases with discriminating functional groups on the tRNA bases have long been known to determine aminoacylation specificity. However, structural and biochemical studies have revealed a second "indirect readout" mechanism that makes an important contribution as well. In indirect readout, the sequence-dependent conformations of tRNA are recognized through protein contacts with the sugar-phosphate backbone and with nonspecific portions of the bases. This mechanism appears to function in single-stranded regions, in canonical A-type duplex segments, and in the complex tertiary core portion of the tRNA. Operation of the indirect mechanism is not exclusive of the direct mechanism, and both are further mediated by induced-fit rearrangements, in which enzyme and tRNA undergo precise conformational changes after formation of an initial encounter complex. The examples of indirect readout in tRNA synthetase complexes extend the concept beyond its traditional application to DNA duplexes and serve as models for the operation of this mechanism in more complex systems such as the ribosome.     

Novel tRNA aminoacylation mechanisms

In nature, ribosomally synthesized proteins can contain at least 22 different amino acids: the 20 common amino acids as well as selenocysteine and pyrrolysine. Each of these amino acids is inserted into proteins codon-specifically via an aminoacyl-transfer RNA (aa-tRNA). In most cases, these aa-tRNAs are biosynthesized directly by a set of highly specific and accurate aminoacyl-tRNA synthetases (aaRSs). However, in some cases aaRSs with relaxed or novel substrate specificities cooperate with other enzymes to generate specific canonical and non-canonical aminoacyl-tRNAs.     

Effect of aminoacylation on tRNA conformation

Translational diffusion coefficients have been simulated for various conformations of tRNA^Phe (yeast) by bead models, in order to analyze data obtained by dynamic light scattering on the free and the aminoacylated form. The 18% increase of the translational diffusion coefficient upon deacylation, reported by Potts et al. (1981), could not be represented by any change of the L-hinge angle, but could only be simulated by a conformation change to an extended form with extensive dissociation of base pairs. Since extensive unpairing is not consistent with evidence accumulated in the literature, the change of the diffusion coefficient must be mainly due to processes other than intramolecular conformational changes.     

A new assay for tRNA aminoacylation kinetics.

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3'-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.     

Inhibition of selenocysteine tRNA[Ser]Sec aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA

GDP dissociation inhibitor (GDI) plays an essential role in regulating the state of bound nucleotides and subcellular localizations of Rab proteins. In our previous study, we showed that OsGDI3 facilitates the recycling of OsRab11 with a help of OsGAP1. In this study, we show that OsGDI3 complement the yeast sec19-1 mutant, a temperature-sensitive allele of the yeast GDI gene, suggesting that OsGDI3 is a functional ortholog of yeast GDI. To obtain further knowledge on the function of OsGDI3, candidate OsGDI3-interacting proteins were identified by yeast two-hybrid screens. OsMAPK2 is one of OsGDI3 interacting proteins from yeast two-hybrid screens and subject to further analysis. A kinase assay showed that the autophosphorylation activity of OsMAPK2 is inhibited by OsGDI3 in vitro. In addition, ectopic expressions of OsGDI3-in Arabidopsis cause reductions at the level of phosphorylated AtMPK in phosphorylation activity. Taken together, OsGDI3 functions as a negative regulator of OsMAPK2 through modulating its kinase activity.     

Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.     

Evidence for a conserved relationship between an acceptor stem and a tRNA for aminoacylation.

The anticodon-independent aminoacylation of RNA hairpin helices that reconstruct tRNA acceptor stems has been demonstrated for at least 10 aminoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA synthetase, the specificity of aminoacylation of the acceptor stem is determined by the U73 nucleotide adjacent to the amino acid attachment site. Because U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cystein enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelixCys based on the acceptor-T psi C stem of yeast tRNACys is a substrate for the E. coli enzyme, and that aminoacylation of this minihelix is dependent on U73. Additionally, we identify two base pairs in the acceptor stem that quantitatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship between the acceptor stem alone and the acceptor stem in the context of a tRNA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cysteine tRNAs is a tertiary base pair at position 15:48 outside of the acceptor stem. Although E. coli tRNACys has an unusual G15:G48 tertiary base pair, yeast tRNACys has a more common G15:C48 that prevents efficient aminoacylation of yeast tRNACys by the E. coli enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian rhythm of tRNA aminoacylation in hepatocyte culture

In the monolayer of rat hepatocytes in vitro, the circadian rhythms were revealed of 3H-leucine incorporation in proteins and in aminoacyl-tRNA(Leu) fraction. The oscillations were mainly synphasic, though coordination between aminoacylation and protein synthesis was not stable in the coarse of time. In cell free system with the excess of ATP and aa-tRNA, the rhythm of 3H-leucine incorporation was also clear. This means, that oscillatory kinetics of the protein synthesis rate is not caused by oscillation of ATP which has been revealed earlier in the hepatocyte monolayer.     

A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme

The ability to recognize tRNA identities is essential to the function of the genetic coding system. In translation aminoacyl-tRNA synthetases (ARSs) recognize the identities of tRNAs and charge them with their cognate amino acids. We show that an in vitro evolved ribozyme can also discriminate between specific tRNAs, and can transfer amino acids to the 3' ends of cognate tRNAs. The ribozyme interacts with both the CCA-3' terminus and the anticodon loop of tRNA(fMet), and its tRNA specificity is controlled by these interactions. This feature allows us to program the selectivity of the ribozyme toward specific tRNAs, and therefore to tailor effective aminoacyl-transfer catalysts. This method potentially provides a means of generating aminoacyl tRNAs that are charged with non-natural amino acids, which could be incorporated into proteins through cell-free translation.     

Two-step aminoacylation of tRNA without channeling in Archaea

Catalysis of sequential reactions is often envisaged to occur by channeling of substrate between enzyme active sites without release into bulk solvent. However, while there are compelling physiological rationales for direct substrate transfer, proper experimental support for the hypothesis is often lacking, particularly for metabolic pathways involving RNA. Here, we apply transient kinetics approaches developed to study channeling in bienzyme complexes to an archaeal protein synthesis pathway featuring the misaminoacylated tRNA intermediate Glu-tRNA^Gln. Experimental and computational elucidation of a kinetic and thermodynamic framework for two-step cognate Gln-tRNA^Gln synthesis demonstrates that the misacylating aminoacyl-tRNA synthetase (GluRS^ND) and the tRNA-dependent amidotransferase (GatDE) function sequentially without channeling. Instead, rapid processing of the misacylated tRNA intermediate by GatDE and preferential elongation factor binding to the cognate Gln-tRNA^Gln together permit accurate protein synthesis without formation of a binary protein-protein complex between GluRS^ND and GatDE. These findings establish an alternate paradigm for protein quality control via two-step pathways for cognate aminoacyl-tRNA formation.     

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.