PROBING THE PROXIMITY OF THE CORE DOMAIN OF AN HIV-1 TAT FRAGMENT IN A TAT-TAR COMPLEX BY AFFINITY CLEAVING

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.