Open AccessFunctional covalent complex between elongation factor Tu and an analog of lysyl-tRNA.
Author(s) -
Arthur E. Johnson,
David L. Miller,
Charles R. Cantor
Publication year - 1978
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.75.7.3075
Subject(s) - ternary complex , ribosome , elongation factor , covalent bond , gtp' , transfer rna , eukaryotic translation elongation factor 1 alpha 1 , ef tu , chemistry , size exclusion chromatography , biochemistry , elongation , biophysics , biology , rna , enzyme , materials science , organic chemistry , ultimate tensile strength , metallurgy , gene
Complex formation between elongation factor Tu, GTP, and Nepsilon-bromoacetyl-Lys-tRNA results in the cross-linking of the protein and the modified Lys-tRNA. The efficiency of affinity labeling is greater than 50%. In the presence of unmodified Lys-tRNA, the amount of crosslinking is greatly decreased. There is no covalent reaction with elongation factor Tu in the absence of complex formation. Substantial purification of the crosslinked ternary complex can be achieved by gel filtration at low Mg2+ concentration and passage through nitrocellulose filters. The crosslinked complex exhibits message-dependent binding to ribosomes which is accompanied by the hydrolysis of the associated GTP, as shown by both filter assays and gel filtration profiles. The crosslinked complex therefore appears to function normally except for its inability to dissociate. These experiments demonstrate that the ternary complex is the true intermediate in the binding of aminoacyl-tRNA to the ribosomes.