Protein Orientation in the Tat-TAR Complex Determined by Psoralen Photocross-linking

Abstract

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a new method based on psoralen photochemistry to identify a specific contact between a fragment of Tat protein (residues 38-72) and TAR RNA. We synthesized a 35-amino acid fragment containing arginine-rich RNA-binding domain of Tat (38-72), and replaced Arg57 with Cys to introduce a unique thiol group (-SH) in our model peptide. A psoralen derivative, which can react with thiol groups, was synthesized and used for specific chemical modification of Cys57-Tat-(38-72). We used this psoralen-Tat conjugate (psoralen-Cys57-Tat-(38-72)) to form a specific complex with TAR RNA. 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 RNA sequencing, which revealed that Cys57 of Tat is close to U31 of TAR RNA. Our results provide high-resolution proximity and orientation information about Tat-TAR complex. 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.