Naslov
Macromolecular complexes of seryl-tRNA synthetases and tRNA(Ser) explored by native electrophoresis

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

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

Izvornik
45 Years of molecular biology in Croatia, 50 years of double helix / Ambriović Ristov, Andreja ; Brozović, Anamaria - Zagreb, 2003

Skup
45 Years of molecular biology in Croatia, 50 years of double helix

Mjesto i datum
Zagreb, Hrvatska, 20.11.2003. - 21.11.2003

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
elektroforeza; seril-tRNA-sintetaza; makromolekulski kompleks; tRNA
(electrophoresis; seryl-tRNA synthetase; macromolecular complex; tRNA)

Sažetak
Aminoacyl-tRNA synthetases aminoacylate tRNAs with cognate amino acids with high specificity, so they are crucial for fidelity of protein biosynthesis. Formation of macromolecular complex between cognate pair of aminoacyl-tRNA synthetase and tRNA is important step in aminoacylation reaction. Macromolecular complexes between tRNASer and homodimeric seryl-tRNA synthetases (SerRS) from yeast and maize organelles were studied by different native electrophoretic methods. Under non-equilibrium conditions, using gel mobility shift assay, only one type of yeast or maize SerRS noncovalent complex could be detected, corresponding to SerRS:tRNASer stoichiometry. Covalent cross-linking has shown that yeast SerRS can bind second tRNA and form SerRS:(tRNASer)2 complex. The stoichiometry of yeast covalent complexes was confirmed by Ferguson analysis. Noncovalent complexes were further analyzed by zone-interference electrophoresis. During zone-interference electrophoresis protein migrates under equilibrium conditions, through a zone of nucleic acid that suppresses dissociation of labile complexes. Thus, less stable complexes can be detected compared to gel mobility shift assay. However, noncovalent complex of two tRNASers bound per yeast SerRS could not be detected even under equilibrium conditions. These findings demonstrate clearly that two binding sites in yeast SerRS dimer have different affinity for tRNASer. In contrast to yeast enzyme, maize organellar SerRS formed two types of macromolecular complexes stable enough to be observed by zone-interference electrophoresis. Maize organellar SerRS:tRNASer complex was detected with tRNASer of different origins (maize mitochondria, E. coli, yeast), but the second complex, probably SerRS:(tRNASer)2, was detected only with organellar and organellar-like (E. coli) tRNASer. This suggests that the second complex may be functionally important 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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

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