Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.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 tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. 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-tRNAPhe-Fl8.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-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.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 A, 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 s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Watson
B S
BS
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Hazlett
T L
TL
Eccleston
J F
JF
Davis
C
C
Jameson
D M
DM
Johnson
A E
AE
D004735:Energy Transfer; D005454:Fluorescence Polarization; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D046911:Macromolecular Substances; D008956:Models, Chemical; D008968:Molecular Conformation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D012346:RNA, Transfer, Amino Acyl; D012235:Rhodamines; C094415:guanosine triphosphate-rhodamine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu