Effect of translocation on topology and conformation of anticodon and D loops of tRNA Phe

Abstract

Steady-state fluorescence and fluorescence anisotropy measurements have been carried out on isolated complexes of fluorescent derivatives of N-AcPhe-tRNA Phe with 70 S ribosomes from Escherichia coli. As a fluorescent probe, proflavine was inserted into either the anticodon loop or the D loop. Upon binding to the A site of poly(U)-programmed ribosomes, the probe in the anticodon loop is highly immobilized and effectively shielded against solvent access in a hydrophobic binding site. Elongation factor G-dependent translocation to the P site does not change any of the fluorescence parameters. These observations indicate that in both sites the environment of the probe with respect to hydrophobicity and shielding against solvent access is rather similar. Moreover, substantial conformational changes of the anticodon loop upon translocation are made unlikely. In contrast to the anticodon loop, the D loop is fully exposed to the solvent in both A and P sites, indicating that the variable region in the middle of the D loop is oriented away from the ribosomal surface. On the other hand, depolarization measurements show that the D loop is strongly immobilized in the A site, possibly by binding interactions of invariant bases of the loop. Upon translocation, the D loop gains considerable flexibility, indicating that in the P site it is neither fixed by contacts with the ribosome nor by intramolecular base-pairing with the T loop. In the absence of poly(U) or in the presence of poly(C), the fluorescence parameters of the probes in the anticodon loop and, more significantly, in the D loop, differ from those observed in the presence of poly(U). These differences are best explained by assuming a codon-induced conformational change of the anticodon loop, which in turn is transmitted to the D loop. When the non-aminoacylated tRNA Phe derivatives are studied, spectroscopic differences as compared to the respective N-AcPhe-tRNA Phe derivatives are observed only for the A site complexes. It appears that the aminoacylation influences the binding of transfer RNA in the A site, but not in the P site.