Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes.
Escherichia coli 16S RNA from 30S ribosomal subunits was isolated, oxidized at the 3' end, and labeled with the thiosemicarbazide derivatives of fluorescein or eosin. Labeled 16S RNA was reconstituted into 30S subunits. They were almost fully active compared to 30S subunits reconstituted from unlabeled 16S RNA by using a poly(uridylic acid)-directed polyphenylalanine synthesis assay. Fluorophores were placed at three different positions of tRNAPhe. E. coli and yeast tRNAPhe were oxidized at the 3' end and labeled with the thiosemicarbazide derivative of fluorescein or with the hydrazide of N-methylanthranilic acid. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin or 1-aminoanthracene. Also, E. coli tRNAPhe was photochemically cross-linked between 4-thiouridine at position 8 and cytidine at position 13. After reduction, this site was used as a fluorescent probe. The labeled tRNAs were bound into the peptidyl site of 70S ribosomes, and then the distances from the fluorophore in the modified tRNA to the fluorophore at the 3' end of 16S RNA were measured by nonradiative energy transfer. Calculations were based on measurements of fluorescence lifetimes. The distances to the 3' end of 16S RNA were found to be as follows: 3' end of tRNA, 67-74 A; cross-linked t RNA, 53-60 A; anticodon loop of tRNA, greater than 61 A.
D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D004735:Energy Transfer; D004926:Escherichia coli / Q000032:analysis*; D010649:Phenylalanine / Q000032:analysis; D012329:RNA, Bacterial / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis*; D012343:RNA, Transfer / Q000032:analysis*; D013050:Spectrometry, Fluorescence
D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D010649:Phenylalanine; D012343:RNA, Transfer