Stoichiometry for the elongation factor Tu.aminoacyl-tRNA complex switches with temperature.
In bacterial protein synthesis binding of an aminoacyl-transferRNA (aa-tRNA) to the ribosomal acceptor site (A-site) is catalyzed by elongation factor Tu (EF-Tu). Two guanosine triphosphates (GTPs) are hydrolyzed on EF-Tu for every bound aa-tRNA. This was rationalized by the notion of an extended ternary complex, consisting of two EF-Tu.GTPs bound to a single aa-tRNA. In this work, we combine fast kinetics with RNase A protection experiments to measure the stoichiometry between EF-Tu.GTP and aa-tRNA at 37 degrees C, where the binding is weak. We find a 2:1 stoichiometry between EF-Tu.GTP and aa-tRNA at 37 degrees C, but at 0 degree C, under otherwise similar conditions, the stoichiometry of the complex is close to 1:1. These results suggest alternative pathways for aa-tRNA binding to ribosomes, since two GTPs are hydrolyzed per peptide bond at both temperatures. At 37 degrees C, aa-tRNA enters the A-site in a pentameric complex with two EF-Tu's on which two GTPs are hydrolyzed in synchrony, when cognate codon-anticodon contact is established. This pentameric model also explains how two GTPs can be hydrolyzed on EF-Tu, without rejection of 50% of the cognate aa-tRNAs in proofreading. At 0 degree C, in contrast, two ordinary ternary complexes may form a pentameric complex on, rather than off, the ribosome. When the two EF-Tu bound GTPs are hydrolyzed, one aa-tRNA enters the A-site, and the other dissociates to the free state.
Bilgin
N
N
Department of Molecular Biology, University of Uppsala, Sweden.
Ehrenberg
M
M
D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism*; D066298:In Vitro Techniques; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature
D012346:RNA, Transfer, Amino Acyl; D006160:Guanosine Triphosphate; D012259:Ribonuclease, Pancreatic; D010444:Peptide Elongation Factor Tu