Gene expression, translation and the behavior of proteins / edited by David M. Prescott, Lester Goldstein.

Date:
1980
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression, translation and the behavior of proteins / edited by David M. Prescott, Lester Goldstein. Source: Wellcome Collection.

    65/520 page 47
    1. The Translational Machinery 47 1. The Prokaryotic Mechanism a. Ternary Complex Formation. EF-Tu forms a binary complex with GTP, which in turn reacts rapidly with aminoacyl-tRNA to form a stable ternary complex with a dissociation constant of about \0~^M (Milierei ai, 1973). The ternary complex may be isolated by gel filtration chromatog¬ raphy or is readily assayed by nitrocellulose filtration that measures a decrease in bound radioactive GTP as EF-Tu • GTP is converted to ter¬ nary complex. The tRNA must be charged, and the aminoacyl group may not be acylated. The initiator tRNA, Met-tRNAj, whether formylated or not, does not bind to EF-Tu • GTP. There appears to be no preference for the positional isomers of aminoacyl-tRNA; both 2'- and 3'-de- oxyadenosine analogues of aminoacyl-tRNA bind equally well (Hecht et ai, 1977). The features of tRNA structure recognized by EF-Tu are not well known, however. Results of X-ray diffraction studies of ternary com¬ plexes are eagerly awaited. Nuclear magnetic resonance spectra of aminoacyl-tRNA as a free molecule or bound in ternary complex are essentially identical, which indicates that extensive changes in the sec¬ ondary and tertiary structures of tRNA do not occur following complex formation (Schulman et ai, 1974). EF-Tu binds to GDP more tightly than to GTP [K^ = 4.9 x 10^ and 3.6 X 10^ M, respectively (Arai et ai, 1974)]. However, the EF- Tu • GDP complex binds to aminoacyl-tRNA with an affinity 10'-fold less than EF-Tu • GTP. EF-Ts plays a role in converting the EF-Tu • GDP complex, which is formed following ribosome binding, to the EF- Tu • GTP complex. The reactions involved are shown in the EF-Tu cycle in Fig. 8. Thus, EF-Tu participates in a cyclic reaction sequence which is driven from equilibrium by GTP hydrolysis on the ribosome. EF-Ts catalyzes the rate of exchange of EF-Tu-bound GDP for GTP. Since the level of EF-Tu in cells is high, approximately equal to that for the sum of tRNA's, most charged tRNA's are present as ternary complexes provided that the energy charge of the cell is sufficiently high. The importance of this fact for proofreading is discussed below in Section IV,B,4. b. Aminoacyl-tRNA Binding to Ribosomes. A ternary complex binds rapidly to ribosomes carrying the appropriate mRNA codon in the A site. Correct binding is followed by GTP hydrolysis to GDP and Pj. EF- Tu • GDP is released from the ribosome and peptide bond formation oc¬ curs (see Fig. 8). The EF-Tu • GDP is then free to be recycled into another ternary complex. When the codon-anticodon sequences are not com¬ plementary, binding and GTP hydrolysis do not readily occur. If the nonhydrolyzable analogue of GTP guanylyl-5'-methylenediphosphonate (GMP-PCP) is substituted, the ternary complex binds, but GTP hydrolysis
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