Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation.

Elongation factor Tu (EF-Tu) binds all elongator aminoacyl-transfer RNAs (aa-tRNAs) for delivery to the ribosome during protein synthesis. Here, we show that EF-Tu binds misacylated tRNAs over a much wider range of affinities than it binds the corresponding correctly acylated tRNAs, suggesting that the protein exhibits considerable specificity for both the amino acid side chain and the tRNA body. The thermodynamic contributions of the amino acid and the tRNA body to the overall binding affinity are independent of each other and compensate for one another when the tRNAs are correctly acylated. Because certain misacylated tRNAs bind EF-Tu significantly more strongly or weakly than cognate aa-tRNAs, EF-Tu may contribute to translational accuracy.

Mapping contacts between Escherichia coli alanyl tRNA synthetase and 2' hydroxyls using a complete tRNA molecule.

A dual-specific derivative of yeast tRNA(Phe) is described whose features facilitate structure-function studies of tRNAs. This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule. A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates. Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.

Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase.

Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.

Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNA(Phe).

The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNA(Phe) mutants was studied. The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNA(Phe) structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNA(Phe). Aminoacylation of tRNA(Phe) anticodon and discriminator base (N(73)) mutants was not affected by pyrophosphatase. Activation of wild-type tRNA(Phe) transcript aminoacylation by inorganic pyrophosphatase was observed only at low Mg(2+) concentrations due to distortion of the tRNA(Phe) structure under these conditions. Our results demonstrate that pyrophosphate dissociation becomes a rate-limiting step of the reaction in yeast phenylalanyl-tRNA synthetase catalyzed aminoacylation of tRNA(Phe) variants with altered tertiary structure. A possible mechanism of pyrophosphate-mediated inhibition of tRNA mutants aminoacylation is discussed.

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.

Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules?

The diadenosine oligophosphates (Ap(n)A) were discovered in the mid-sixties in the course of studies on aminoacyl-tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap(n)A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap(n)A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon-inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2-5A synthetase, also interferon-inducible, priming the synthesis of 2' adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit.Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap(n)A family is potentially a novel class of signal-transducing molecules whose functions are yet to be determined.

Compartmentalization of certain components of the protein synthesis apparatus in mammalian cells.

A comparative study on the localization of free cytosolic tryptophanyl-tRNA synthetase (TrpRS) and several components of the multi-aminoacyl-tRNA synthetase (ARS) complex (glutamyl-prolyl-tRNA synthetase (GluProRS), arginyl-tRNA synthetase (ArgRS)), and two non-synthetase polypeptides p38 and p43 has been carried out on ultrathin sections of cultured rabbit kidney cells by the immunogold technique using monoclonal antibodies raised against appropriate polypeptides. It has been shown that GluProRS, ArgRS and p38 polypeptide are distributed in the cells similarly to TrpRS and are located mainly in the vicinity of ribosomes. A smaller but significant portion of these proteins has been observed in the nuclei in the diffuse chromatin regions and in the vicinity of interchromatin granules. On the contrary, the main part of p43 protein was found in the cell nuclei; this indicates that this protein may exist in the cell separately from the cytoplasmic multi-ARS complex. Our results argue in favor of compartmentalization of both free ARS (such as TrpRS) and the multi-ARS complex in the vicinity of ribosomes. At the same time, the detection of some ARS in the diffuse chromatin regions in the nucleus implies that these enzymes may exhibit some non-canonical functions in addition to their role in protein synthesis.

Anticodon-dependent aminoacylation of RNA minisubstrate by lysyl-tRNA synthetase.

Specific inhibition of mammalian lysyl-tRNA synthetase by polyU is shown. Inhibition of the enzyme is dependent on the length of the oligonucleotide, since oligoU molecules with a length of less than 8 residues do not inhibit the aminoacylation, whilst the effect of oligoU molecules with a length of about 30 residues is the same as that of polyU. Inhibition is a result of recognition by the enzyme of the tRNALys anticodon sequence (UUU) coded by polyU. Aminoacylation of the oligoU molecule with attached CCA sequence (G(U)20-CCA) by yeast and mammalian lysyl-tRNA synthetases is demonstrated.

Crucial role of pyrophosphate in the aminoacylation of E. coli tRNA(Phe) by yeast phenylalanyl-tRNA synthetase.

Rapid inactivation of the yeast phenylalanyl-tRNA synthetase in the course of aminoacylation of the heterologous E. coli tRNA(Phe) is observed. This inactivation occurs due to the formation of the tight complex of the enzyme with the pyrophosphate formed during the aminoacylation reaction. This complex is shown to be the normal intermediate of the reaction. Possible inactivation mechanism and correlation between structural differences of yeast and E. coli tRNAs(Phe) with the changes in the enzymatic mechanism of aminoacylation are discussed.

Purification and properties of a high-molecular-mass complex between Val-tRNA synthetase and the heavy form of elongation factor 1 from mammalian cells.

In extracts of various mammalian tissues obtained in the presence of protease inhibitors Val-tRNA synthetase exists exclusively as a complex with a molecular mass of about 800 kDa. This complex was purified by gel filtration and two HPLC steps and contained five different polypeptides with molecular masses of 140, 50, 50, 40 and 30 kDa. The complex seems to have no tissue or species specificity, because preparations with identical polypeptide composition were obtained by the same method from rabbit liver and reticulocytes, and rat and beef liver. Four low-molecular-mass polypeptides were identified by two-dimensional electrophoresis as subunits of the heavy form of elongation factor 1 (EF-1H). The complex possesses the activity of EF-1 in the poly(U)-directed translation system, indicating that EF-1H is an integral part of the complex. Gel filtration of the tissue extracts reveals three different peaks of EF-1 activity, corresponding to EF-1 alpha, EF-1H and the high-molecular-mass complex of Val-tRNA synthetase and EF-1H. All activity of Val-tRNA synthetase and about 25% of EF-1 activity are associated with the complex. Different forms of EF-1 revealed no significant differences in the nucleotide-binding properties, but the complex of Val-tRNA synthetase with EF-1H was 10 times more active in the poly(U)-directed binding of Phe-tRNAPhe to ribosomes than EF-1H. These results strongly suggest that the complex of Val-tRNA synthetase with EF-1H is a novel functionally active individual form of EF-1.

Monoclonal antibodies to the components of the high-molecular-mass aminoacyl-tRNA synthetase complex.

Six monoclonal antibodies to the components of the rabbit multienzyme aminoacyl-tRNA synthetase complex were generated and characterised. Two, F7 and F31 were directed against arginyl-tRNA synthetase, two, F8 and F25 against glutamyl-tRNA synthetase, and two, F6 and F12 recognised 38 and 43 kDa polypeptides, respectively. All antibodies were species-specific and failed to affect the activity of the respective enzymes.

Adaptation of an HPLC system for transient-state enzyme kinetic experiments: pulse-flow method.

The components of an HPLC system can be easily reassembled into an instrument for transient-state enzyme kinetic experiments based on the continuous-flow technique. The scheme of reassembly, operation and data evaluation is described in detail for the Pharmacia FPLC system. The working quality of the suggested scheme was tested using a well-studied process of bacterial beta-galactosidase activation by Mg2+ ions. The found parameters were in a good agreement with the literature data. The pulse-flow mode was developed for the delivery of working solutions to reduce the required quantities of enzyme and/or other reaction components. The work presents new approach to HPLC modules as building blocks for sophisticated liquid flow reactors, such as automatic solid phase synthesizers, sequencers and so on.

High-performance ion-exchange chromatography of oligoribonucleotides using linear and hyperbolic salt gradients.

A method for separation and chain length determination of oligo- and polynucleotides by high-performance anion-exchange chromatography was developed, which allows resolution of individual fragments according to their chain length n, up to n approximately 10 by linear gradient of sodium chloride and up to n approximately 30 by an hyperbolic gradient of this salt. The hyperbolic relationship between n and the salt concentration at which elution of the fragment occurs allows determination of the degree of polymerization of oligo- and polynucleotides with unknown n. The method proposed can be used for estimation of the effective charge of nucleic acids with complex structure.

Mammalian valyl-tRNA synthetase forms a complex with the first elongation factor.

The high-molecular-mass form of valyl-tRNA synthetase is associated with the first elongation factor activity. It includes two polypeptides of about 50 kDa and two others of 40 and 30 kDa, identified as alpha, beta, gamma and delta subunits of eEF-1H. The complex of valyl-tRNA synthetase with eEF-1H is suggested to be a novel form of the first elongation factor.

Purification of valyl-tRNA synthetase high-molecular-mass complex from rabbit liver.

A high-molecular-mass complex containing valyl-tRNA synthetase has been purified to homogeneity from rabbit liver. The molecular mass of the complex is about 800 kDa. The complex consists of four polypeptides of 130, 50, 40 and 30 kDa.