The conformation of the anticodon loop of yeast tRNAPhe in solution and on ribosomes

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Abstract
The Y‐base of yeast tRNAPhe was replaced by the fluorophores 1‐aminoanthracene or proflavine to yield derivatives which are active in all of the reactions of peptide elongation on reticulocyte ribosomes. The relatively long lifetime, higher quantum yield, and environmental sensitivity of 1‐aminoanthracene make it a particulary useful adjunct to the Y‐base in studying conformational changes in the anticodon region. The absorption and emission spectra of 1‐aminoanthracene in tRNA in solutions in which it is active in peptide synthesis indicate that the probe is in a hydrophobic environment, apparently provided by stacking with the adjacent bases in the anticodon loop. The proflavine derivative, tRNA, was employed in iodide quenching, D2O enhancement, and fluorescence depolarization experiments. The results indicate that the fluorophore in partially but not completely protected from the solvent. Anisotropy studies indicate that in solutions approximating those which support peptide synthesis on ribosomes, the probes have significant but restricted flexibility within the anticodon loop. Considered with nmr data and Y‐base fluorescence from crystals of tRNA, the results indicate that the solution and crystal structures of tRNAPhe are very similar. In turn, fluorescene from modified tRNAPhe bound to ribosomes is similar to that observed in solution. It is of special significance for future experiments involving nonradiative energy transfer that these probles adjacent to the anticodon retain independent flexibility when bound to ribosomes with poly(U). The tRNAPhe itself appears to be held rigidly on the ribosomes. It is concluded that within the limits dictated by the position and sensitivity of the probes used in this study, the mechanism of tRNAPhe binding to ribosomes and the movement of tRNA and mRNA during the translocation steps of peptide synthesis can be interpreted in terms of the well‐defined crystal structure of tRNAPhe.