Naslov
STABILNOST I STEHIOMETRIJA KOMPLEKSA SERIL-tRNA SINTETAZA I tRNASer
(STABILITY AND STOICHIOMETRY OF COMPLEXES BETWEEN SERYL-tRNA SYNTHETASES AND tRNASer)

Autori
Močibob, Marko ; Gruić-Sovulj, Ita ; Weygand-Đurašević, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
XVIII. hrvatski skup kemičara i kemijskih inženjera / Zrnčević, Stanka - Zagreb : Hrvatsko društvo kemijskih inženjera i tehnologa (HDKI), 2003

Skup
XVIII. hrvatski skup kemičara i kemijskih inženjera

Mjesto i datum
Zagreb, Hrvatska, 16.02.2003. - 19.02.2003

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
aminoacil-tRNA-sintetaze; tRNA; nekovalentni kompleksi
(aminoacyl-tRNA-synthetases; tRNA; noncovalent complexes)

Sažetak
Formation of noncovalent complex between cognate pair of aminoacyl-tRNA synthetase and tRNA is important step in the aminoacylation reaction. In present work complexes between tRNASer and homodimeric seryl-tRNA synthetases (SerRS) from yeast cytosol and maize organelles were studied by different native electrophoretic methods. Experiments were conducted under equilibrium (zone-interference electrophoresis) and non-equilibrium (gel mobility shift assay) conditions. Only the noncovalent complex with stoichiometry of one tRNASer per dimeric yeast SerRS, SerRS:tRNASer, was stable enough to be detected using mobility shift assay. The same result was obtained with maize enzyme. Covalent cross-linking has shown that yeast SerRS binds second tRNASer, although with significantly decreased affinity. The stoichiometry of yeast covalent complexes was confirmed by Ferguson analysis. Noncovalent complex of two tRNASers bound per SerRS dimer was not detected even by zone-interference electrophoresis. During zone-interference electrophoresis protein migrates under equilibrium conditions, through a zone of nucleic acid that suppresses dissociation. This enables detection of less stable complexes compared to gel mobility shift assay. Therefore, presented results strongly confirm different affinity of two binding sites for tRNASer. In contrast to yeast enzyme, maize organellar SerRS formed two types of noncovalent complexes sufficiently stable for the detection by zone-interference electrophoresis. SerRS:tRNASer complex was detected with maize mitochondrial, E. coli and yeast tRNASer. The second complex, presumably SerRS:(tRNASer)2, could be detected only with cognate or cognate-like (E. coli) tRNASer, suggesting that it may be catalytically relevant complex in vivo. Both methods were used to explore the influence of Mg2+ ions on yeast SerRS:tRNASer complex stability. Zone-interference electrophoresis revealed that SerRS:tRNASer can be formed in the absence of Mg2+ ions, but it is not kinetically stable to be detected by gel mobility shift assay. Electrophoresis performed in the presence and absence of Mg2+ ions implies that Mg2+ ions mainly affect kinetic, not thermodynamic, properties of yeast SerRS:tRNASer complex. Presented results show that the comparison of the equilibrium and non-equilibrium electrophoretic assays is useful in the analysis of stability, stoichiometry and kinetic properties of complexes between seryl-tRNA synthetases and tRNASer.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA