Codon-dependent rearrangement of the three-dimensional structure of phenylalanine tRNA, exposing the T-ψ-C-G sequence for binding to the 50S ribosomal subunit

Abstract

Codon-anticodon interaction induces an allosteric rearrangement of the 3-dimensional structure of Phe-tRNAPhe [Escherichia coli] that exposes the T-.psi.-C-G sequence for binding to the C-G-A-A sequence of the 5S rRNA within the 50S ribosomal subunit. The conformational change in the tRNAPhe structure was followed by the binding of C-G-[3H]A-[3H]A to the T-.psi.-C-G sequence, as measured by equilibrium dialysis at 10 mM Mg2+. C-G-A-A (14 pmol) was bound to tRNAPhe in the complete system containing elongation factor Tu .cntdot. GTP .cntdot. Phe-tRNA .cntdot. (uridylyl-3'',5'')7-uridine .cntdot. 30S ribosomes (100 pmol). At a Mg2+ concentration lower than 5 mM the rearrangement was dependent on elongation factor-Tu, whereas GTP could be replaced by guanylyl imidodiphosphonate. In the absence of elongation factor-Tu .cntdot. GTP a sigmoidal C-G-A-A binding curve with respect to Mg2+ concentration was obtained, showing half-saturation at 6 mM Mg2+. To achieve the change in the tRNAPhe structure in the absence of 30S ribosomes, a 2-fold higher concentration of (uridylyl-3'',5'')7-uridine had to be used. A sigmoidal curve was obtained again when the Mg2+ dependence of the C-G-A-A binding was followed, with 12 pmol of C-G-A-A being bound to 200 pmol of Phe-tRNA. Since T-.psi.-C-G exposure should influence the binding of Phe-tRNA to 70S ribosomes, Phe-tRNA binding to 70S ribosomes was examined. In the nonenzymatic binding (i.e., no elongation factor-Tu .cntdot. GTP) of Phe-tRNA a sigmoidal Mg2+ dependence was found, whereas the enzymatic binding (elongation factor-Tu.cntdot.GTP present) showed a hyperbolic curve. With 30S ribosomes as controls, only hyperbolic binding curves were found. The Mg2+ dependence of aminoacyl-tRNA binding thus reflects the rearrangement of the tRNA structure.