The equilibrium binding of a highly fluorescent derivative of yeast tRNAPhe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 109 m-1 and (3.4 ± 0.2) × 107 m-1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.
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