Ribosome binding by tRNAs with fluorescent labeled 3′ termini

Yeast and E. coli tRNAphesamples were oxidized and labeled at the 3′ end with dansy1 hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8°C. Two binding sites with binding constants of about 1 × 10°9 M−1 (P) and 3×107 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 showed that the distance from the 3′ end of tRNAphe in the P site to a fluorescein derivative of erythromycin is 23 Å. This supports in vitro studies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 Å between the 3′ ends of 2 tRNAs bound simultaneously 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.