Novel complexes of mammalian translation elongation factor eEF1A.GDP with uncharged tRNA and aminoacyl-tRNA synthetase. Implications for tRNA channeling.
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, B.S. & El'skaya, A.V. (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, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (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), e.g. 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 (Kd = 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 of eEF1A.GDP with tRNA and ARS in the channeled elongation cycle is discussed.
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D004355:Drug Stability; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D010441:Peptide Chain Elongation, Translational; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D020349:Surface Plasmon Resonance
D046911:Macromolecular Substances; D020648:Peptide Elongation Factor 1; D012360:RNA, Transfer, Phe; D006153:Guanosine Diphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase