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N S Kholod

Publications and source records attributed to N S Kholod.

6 recordsLinked to original sources
PubMed1999

Dimer formation by tRNAs.

Available data on formation of dimers and other oligomers by "mature" tRNAs and the transcripts of their genes show that the dimers may be formed in vitro during the purification of individual tRNAs as well as on heating and subsequent cooling of tRNA preparations. The functional activity of dimers varies for different tRNAs. Structural analysis of tRNAs that are able or unable to form dimers suggests the importance of the hairpin melting temperature for dimer formation. Pathways of dimer formation and specific structural features of tRNA dimers are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration.

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

Acylation↗
PubMedDate not supplied

[A new method to measure the functional activity of class-1 translation termination factor eRF1].

Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene].

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

Animals↗