title: Ribosome binding by tRNAs with fluorescent labeled 3' termini.
abstract: Yeast and E. coli tRNAPhe samples were oxidized and labeled at the 3' end with dansyl hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8 degrees C. Two binding sites with binding constants of about 1 X 10(9) M-1 (P) and 3 X 10(7) M-1 (A) were determined for the yeast tRNAPhe derivatives. With E. coli tRNAPhe the A site affinity is similar to yeast tRNAPhe but the P site affinity is 5-fold weaker. Singlet-singlet energy transfer showd that the distance from the 3' end of tRNAPhe in the P site to a fluorescein derivative of erythromycin is 23 A. This supports in vitro studies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 A between the 3' ends of 2 tRNAs bound simulatneously on the ribosome was also measured. This long distance may mean that the deacylated fluorescent tRNA binds to the A site in an orientation like that in the stringent response rather than in protein synthesis.
mesh_terms: D001665:Binding Sites; D003619:Dansyl Compounds; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006834:Hydrazines; D010649:Phenylalanine; D012329:RNA, Bacterial / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D003619:Dansyl Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006834:Hydrazines; D012329:RNA, Bacterial; D012331:RNA, Fungal; C027913:dansyl hydrazine; D010649:Phenylalanine; C027912:fluorescein thiosemicarbazide; D012343:RNA, Transfer