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 GTP 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-thiouridine 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. Formation of the Phe-tRNA(Phe)-Fl(8) . EF-Tu . GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical 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 excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 Angstrom, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment 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 limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex. When coupled with previous cross-linking data that localized 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 to position the s(4)U-8 base far from the GTP ribose.