Changes in aminoacyl transfer ribonucleic acid conformation upon association with elongation factor Tu-guanosine 5'-triphosphate. Fluorescence studies of ternary complex conformation and topology

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Abstract
The association of [Escherichia coli] aminoacyl-tRNA (aa-tRNA) with elongation factor Tu.cntdot.GTP to form an aa-tRNA.cntdot.EF-Tu.cntdot.GTP ternary complex was investigated by using 2 different fluorescent probes, both of which monitored structural changes near the juncture of the 2 arms of the L-shaped tRNA. Aminoacylation of tRNAPhe-F8, a functionally active analog of tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base, did not cause a change in the fluorescence emission intensity. However, when EF-Tu.cntdot.GTP bound to Phe-tRNAPhe-F8, the emission intensity increased by .apprx. 30%, depending upon the solvent conditions. About half of this increase in fluorescence was due to an increase in the molar absorptivity of the fluorescein dye. Ternary complex formation did not alter the rate of I- ion quenching of the Phe-tRNAPhe-F8 fluorescence. Since solvent access to fluorescein was not reduced when EF-Tu.cntdot.GTP was bound to Phe-tRNAPhe-F8, the fluorescence intensity change noted above was not caused by a direct interaction between fluorescein and EF-Tu. Instead, the binding of EF-Tu.cntdot.GTP to the aa-tRNA resulted in a conformational change in the aa-tRNA near s4U-8. Ternary complex formation also altered the nature of the single strong binding site for ethidium in unfractionated and unmodified aa-tRNA. However, ethidium binding to its strong site was not blocked. Only the acceptor-T.PSI.C [.PSI. = pseudouridine] arm of aa-tRNA interacts directly with EF-Tu.cntdot.GTP and the anticodon-D arm is available for direct interaction with the ribosome during recogition. EF-Tu may facilitate protein biosynthesis by ensuring that every aa-tRNA is in a particular (possibly the same) conformation prior to initiation of the recognition process at the ribosomal complex.