Interactions of yeast tRNAPhe with ribosomes from yeast and Escherichia coli. A fluorescence spectroscopic study.
The interaction of ethidium-labeled tRNAPhe from yeast with ribosomes from yeast and Escherichia coli was studied by stead-state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon-independent formation of a tRNA-ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non-labeled tRNAPhe showed that the labeled tRNAPheEtd was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon-anticodon interaction on the ribosome suggests a conformational change of the ribosome-bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TpsiC loop.
D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000378:metabolism*; D004996:Ethidium; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010649:Phenylalanine; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013045:Species Specificity; D013050:Spectrometry, Fluorescence
D010649:Phenylalanine; D012343:RNA, Transfer; D004996:Ethidium; D008274:Magnesium