MACROMOLECULAR ARRANGEMENT IN THE AMINOACYL-TRANSFER-RNA-CENTER-DOT-ELONGATION FACTOR TU-CENTER-DOT-GTP TERNARY COMPLEX - A FLUORESCENCE ENERGY-TRANSFER STUDY

The distance between the corner of the L-shaped transfer RNA and the G TP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA . EF-Tu . GTP ternary complex was measured using fluorescence energy transfer . The donor dye, fluorescein (Fl), was attached covalently to the 4-th iouridine base at position 8 of tRNA(Phe), and aminoacylation yielded Phe-tRNA(Phe)-Fl(8). The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. F ormation of the Phe-tRNA(Phe)-Fl(8) . EF-Tu . GTP-Rh ternary complex w as verified both by EF-Tu protection of the aminoacyl bond from chemic al hydrolysis and by an EF-Tu . GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNA(Phe)-Fl(8) . EF-Tu . GTP ternary complex than in the Phe-tRNA(Phe)-Fl(8) . EF-Tu . GTP-Rh ternary complex. These spectral differences disappeared when e xcess GTP was added to replace GTP-Rh in the latter ternary complex, t hereby showing that excited-state energy was transferred from fluoresc ein to rhodamine in the ternary complex. The efficiency of singlet-sin glet energy transfer was low (10-12%), corresponding to a distance bet ween the donor and acceptor dyes in the ternary complex of 70 +/- 7 An gstrom, where the indicated uncertainty reflects the uncertainty in dy e orientation. After correction for the lengths of the probe attachmen t tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s (4)U are separated by a minimum of 49 Angstrom. This large distance li mits the possible arrangements of the EF-Tu and the tRNA in the ternar y complex. When coupled with previous cross-linking data that localize d the aminoacyl end of the tRNA acceptor arm near His66, the acceptor arm must extend from near His66 away from the GTP binding site so as t o position the s(4)U-8 base far from the GTP ribose.