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.

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.

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.

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.