The identification of the determinants of the cyclic, sequential binding of elongation factors tu and g to the ribosome.
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.
Yu
Huijun
H
Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
Chan
Yuen-Ling
YL
Wool
Ira G
IG
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*
D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D010444:Peptide Elongation Factor Tu