tailieunhanh - Báo cáo Y học: Novel complexes of mammalian translation elongation factor eEF1AÆGDP with uncharged tRNA and aminoacyl-tRNA synthetase

Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNAduring protein synthesis [Negrutskii, ’skaya, . (1998)Prog. Nucleic Acids Res. Mol. –78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl-tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, ., Negrutskii, .,. | Eur. J. Biochem. 269 4811-4818 2002 FEBS 2002 doi Novel complexes of mammalian translation elongation factor eEF1A-GDP with uncharged tRNA and aminoacyl-tRNA synthetase Implications for tRNA channeling Zoya M. Petrushenko Tatyana V. Budkevich Vyacheslav F. Shalak Boris S. Negrutskii and Anna V. El skaya Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine Kiev Ukraine Multimolecular complexes involving the eukaryotic elongation factor 1A eEF1A have been suggested to play an important role in the channeling vectorial transfer of tRNA during protein synthesis Negrutskii . El skaya . 1998 Prog. Nucleic Acids Res. Mol. Biol. 60 47-78 Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl-tRNA the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA Petrushenko . Negrutskii . Ladokhin . Budkevich . Shalak . El skaya . 1997 FEBS Lett. 407 13-17 . The eEF1A-GDP-tRNA complex has been hypothesized to interact with aminoacyl-tRNA synthetase ARS resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the eEF1A-GDP-tRNA complex with phenylalanyl-tRNA synthetase PheRS . the formation of the above quaternary complex detected by the gel-retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the eEF1A-GDP-tRNA and eEF1A-GDP-tRNAPhe-PheRS complexes were found to be 20 nM and 9 nM respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe Ki 21 nM . However the addition of tRNAPhe accelerated eEF1A-GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes .