CHEMICAL CONVERSION OF A TRANSACTIVATION RESPONSIVE RNA-BINDING FRAGMENT OF HIV-1 TAT PROTEIN INTO A SITE-SPECIFIC CROSS-LINKING AGENT

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive (TAR) region of RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. We have devised a new method based on psoralen photochemistry to identify a specific contact between a fragment of Tat protein (residues 42-72) and TAR RNA. We synthesized a 30-amino acid fragment containing the arginine-rich RNA-binding domain of Tat(42-72) and chemically attached a psoralen at the amino terminus. Upon near ultraviolet irradiation (360 nm), this synthetic psoralen-peptide cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified, and the cross-link site on TAR RNA was determined by chemical and primer extension analyses. Our results show that the amino terminus of Tat(42-72) contacts, or is close to, uridine 42 in the lower stem of TAR RNA. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.