MACROMOLECULAR ARRANGEMENT IN THE AMINOACYL-TRANSFER-RNA-CENTER-DOT-ELONGATION FACTOR TU-CENTER-DOT-GTP TERNARY COMPLEX - A FLUORESCENCE ENERGY-TRANSFER STUDY
Bs. Watson et al., MACROMOLECULAR ARRANGEMENT IN THE AMINOACYL-TRANSFER-RNA-CENTER-DOT-ELONGATION FACTOR TU-CENTER-DOT-GTP TERNARY COMPLEX - A FLUORESCENCE ENERGY-TRANSFER STUDY, Biochemistry, 34(24), 1995, pp. 7904-7912
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.