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 structure located at the 5'-end of all mRNAs. We have used a site-directed RNA-cleaving strategy to determine the neighborhood of the core domain of a Tat fragment in the Tat-TAR complex. We synthesized a 35-amino acid fragment containing arginine-rich RNA-binding domain of Tat(38-72) and attached an EDTA analog to its amino terminus. A derivative of (p-aminobenzyl)-EDTA tetra-tert-butyl ester was synthesized and attached to the amino terminus of the Tat peptide by standard peptide coupling methods. Cleavage from the resin and deprotection of the peptide were carried out in trifluoroacetic acid which also generated unprotected metal binding EDTA moieties. We used this EDTA-Tat conjugate to form a specific complex with TAR RNA. This sequence-specific RNA-binding peptide was converted into a sequence-specific RNA-cleaving peptide by 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 A35), and at the strand opposite the bulge (U40 and C41). These results demonstrate that, in the three-dimensional structure of the Tat-TAR complex, the Phe38 of Tat(38-72) is located in the proximity of the bulge region and two nucleotides from the loop sequence.
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Department of Pharmacology, Robert Wood Johnson (Rutgers) Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
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D000595:Amino Acid Sequence; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus