Initial binding of the elongation factor Tu.GTP.aminoacyl-tRNA complex preceding codon recognition on the ribosome.
The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time-resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives. The complex is labile, with rate constants of 6 x 10(7) M-1 s-1 and 24 s-1 (20 degrees C, 10 mM Mg2+) for binding and dissociation, respectively. Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction. While a cognate ternary complex proceeds form initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases. Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.
Rodnina
M V
MV
Institut für Molekularbiologie, Universität Witten/Herdecke, Witten, Federal Republic of Germany.
Pape
T
T
Fricke
R
R
Kuhn
L
L
Wintermeyer
W
W
D003062:Codon / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008954:Models, Biological; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D003062:Codon; D005456:Fluorescent Dyes; D012346:RNA, Transfer, Amino Acyl; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu