I. Huq et Tm. Rana, PROBING THE PROXIMITY OF THE CORE DOMAIN OF AN HIV-1 TAT FRAGMENT IN A TAT-TAR COMPLEX BY AFFINITY CLEAVING, Biochemistry, 36(41), 1997, pp. 12592-12599
Transactivation of human immunodeficiency virus (HIV) gene expression
depends upon the interaction of the viral regulatory protein Tat with
the transactivation responsive region (TAR) RNA, a 59-base stem-loop s
tructure located at the 5'-end of all mRNAs. We have used a site-direc
ted RNA-cleaving strategy to determine the neighborhood of the core do
main of a Tar fragment in the Tar-TAR complex. We synthesized a 35-ami
no acid fragment containing arginine-rich RNA-binding domain of Tat(38
-72) and attached an EDTA analog to its amino terminus. A derivative o
f (p-aminobenzyl)-EDTA tetra-tert-butyl ester was synthesized and atta
ched to the amino terminus of the Tat peptide by standard peptide coup
ling methods, Cleavage from the resin and deprotection of the peptide
were carried out in trifluoroacetic acid which also generated unprotec
ted metal binding EDTA moieties. We used this EDTA-Tat conjugate to fo
rm a specific complex with TAR RNA, This sequence-specific RNA-binding
peptide was converted into a sequence-specific RNA-cleaving peptide b
y the addition of Fe(II) salt, ascorbate, and H2O2. Hydroxyl radicals
generated from the tethered Fe(II) cleaved the TAR RNA backbone in two
localized regions. Site-specific cleavage of TAR RNA was observed at
the bulge residues (U23, C24, and U25), in the loop region (G34 and A3
5), and at the strand opposite the bulge (U40 and C41). These results
demonstrate that, in the three-dimensional structure of the Tat-TAR co
mplex, the Phe38 of Tat(38-72) is located in the proximity of the bulg
e region and two nucleotides from the loop sequence.