1. The identification of the determinants of the cyclic, sequential binding of elongation factors tu and g to the ribosome.
- Author
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Yu H, Chan YL, and Wool IG
- Subjects
- Base Sequence, Binding Sites, Escherichia coli chemistry, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Protein Binding, RNA, Bacterial chemistry, RNA, Bacterial metabolism, RNA, Ribosomal, 23S chemistry, RNA, Ribosomal, 23S metabolism, Ribosome Subunits, Large, Bacterial chemistry, Escherichia coli metabolism, Peptide Elongation Factor G metabolism, Peptide Elongation Factor Tu metabolism, Ribosomes chemistry, Ribosomes metabolism
- Abstract
Experiments dedicated to gaining an understanding of the mechanism underlying the orderly, sequential association of elongation factor Tu (EF-Tu) and elongation factor G (EF-G) with the ribosome during protein synthesis were undertaken. The binding of one EF is always followed by the binding of the other, despite the two sharing the same-or a largely overlapping-site and despite the two having isosteric structures. Aminoacyl-tRNA, peptidyl-tRNA, and deacylated-tRNA were bound in various combinations to the A-site, P-site, or E-site of ribosomes, and their effect on conformation in the peptidyl transferase center, the GTPase-associated center, and the sarcin/ricin domain (SRD) was determined. In addition, the effect of the ribosome complexes on sensitivity to the ribotoxins sarcin and pokeweed antiviral protein and on the binding of EF-G*GTP were assessed. The results support the following conclusions: the EF-Tu ternary complex binds to the A-site whenever it is vacant and the P-site has peptidyl-tRNA; and association of the EF-Tu ternary complex is prevented, simply by steric hindrance, when the A-site is occupied by peptidyl-tRNA. On the other hand, the affinity of the ribosome for EF-G*GTP is increased when peptidyl-tRNA is in the A-site, and the increase is the result of a conformational change in the SRD. We propose that peptidyl-tRNA in the A-site is an effector that initiates a series of changes in tertiary interactions between nucleotides in the peptidyl transferase center, the SRD, and the GTPase-associated center of 23S rRNA; and that the signal, transmitted through a transduction pathway, informs the ribosome of the position of peptidyl-tRNA and leads to a conformational change in the SRD that favors binding of EF-G.
- Published
- 2009
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