E. coli unfractionated tRNA and tRNA phe both contain a single strong ethidium binding site. Singlet-singlet energy transfer has been used to measure the distance between this site and dansyl hydrazine covalently attached to the 3' end of the tRNAs. The distance obtained is between 33 and 40 A for both samples. This is completely consistent with results from earlier NMR studies which placed the single, strong ethidium binding site of yeast tRNAphe between base pairs 6 and 7 on the aminoacyl stem. From the known tertiary structure of tRNAphe it is possible to rationalize the unusual affinity of this site and its likely existence in all tRNAs.

Micrococcus lysodeikticus DNA; CT-DNA, calf thymus DNA; RDV-RNA, rice dwarf virus RNA; CPV-RNA, cytoplasmic polyhedrosis virus RNA.

The intrinsic fluorescence of the Wye base was used to study the conformational change of the anticodon loop of yeast tRNAPhe brought about by the addition of magnesium. The fluorescence emission and excitation spectra show dramatic changes as magnesium is added to the solution. The rotational relaxation time changes from 6 nsec without added magnesium to 33 nsec with 10 mM magnesium at an ionic strength of 0.1 M. Stern-Volmer quenching by iodide or iodoethanol shows greater access of the base to the quencher with no added magnesium. A plausible interpretation of this data is that the base stack of the anticodon loop is altered by tilting or twisting the Wye base with respect to the adjacent bases and the base becomes parallel to its neighbors upon the addition of magnesium.

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.

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