Codon-dependent rearrangement of the three-dimensional structure of phenylalanine tRNA, exposing the T-psi-C-G sequence for binding to the 50S ribosomal subunit.
Codon-anticodon interaction induces an allosteric rearrangement of the three-dimensional structure of Phe-tRNAPhe 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-GTP-Phe-tRNA-(uridylyl-3',5')7-uridine-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-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 twofold 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-GTP) of Phe-tRNA a sigmoidal Mg2+ dependence was found, whereas the "enzymatic" binding (elongation factor-Tu-GTP present) showed a hyperbolic curve. With 30S ribosomes as controls, only hyperbolic binding curves were found. The Mg2+ dependence of AA-tRNA binding thus reflects the rearrangement of the tRNA structure.
D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D010649:Phenylalanine; D012333:RNA, Messenger; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D003062:Codon; D012333:RNA, Messenger; D010649:Phenylalanine; D012343:RNA, Transfer; D008274:Magnesium