PMID:7827027
| Citation |
Bilgin, N and Ehrenberg, M (1995) Stoichiometry for the elongation factor Tu.aminoacyl-tRNA complex switches with temperature. Biochemistry 34:715-9 |
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| Abstract |
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. |
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| Keywords |
Escherichia coli; Guanosine Triphosphate/metabolism; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu/metabolism; RNA, Transfer, Amino Acyl/metabolism; Ribonuclease, Pancreatic/metabolism; Ribosomes/metabolism; Temperature |
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