TRANSIENT CONFORMATIONAL STATES OF AMINOACYL-TRANSFER-RNA DURING RIBOSOME BINDING CATALYZED BY ELONGATION-FACTOR TU

Conformational transitions of Phe-tRNA(Phe) that take place during elo ngation factor Tu (EF-Tu)-dependent binding to the A site of Escherich ia coil ribosomes were followed by transient fluorescence measurements . The fluorescence signal of proflavin replacing dihydrouracil at posi tion 16 or 17 in yeast tRNA(Phe) was utilized to monitor changes of th e conformation of the D loop. The ternary complex EF-Tu.GTP.Phe-tRNA(P he)(Prf16/17) was purified by gel filtration. Upon binding of the comp lex to the A site of poly(U)-programmed, P-site-blocked ribosomes, the fluorescence changes in several steps. First, the rapid formation of an initial complex gives rise to a small fluorescence increase. Subseq uent condon-anticodon recognition leads to a conformational rearrangem ent of the D loop of the tRNA that is reflected In a major fluorescenc e increase. Fluorescence-quenching data indicate an unfolding of the D loop in this state. The latter conformational state is short-lived, a nd the aminoacyl-tRNA refolds during the following rearrangement that occurs after GTP hydrolysis and accompanies the release of the aminoac yl-tRNA from EF-Tu.GDP and/or its accommodation in the A site. Further experiments show that the status df the P site influences the binding to the A site in that the two rearrangement steps are slowed down whe n the P site is unoccupied and even more so when it is occupied with t he near-cognate tRNA(Leu2) In contrast, the occupancy of the E site ha s no influence on A-site binding, and vice versa, thus excluding any c oupling between the two sites.