Proximity of a Tat peptide to the HIV-1 TAR RNA loop region determined by site-specific photo-cross-linking

Abstract

Transcriptional regulation in 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'-ends of all HIV-1 mRNAs. We have used a site-specific cross-linking method based on 4-thio-uracil (4-thioU) photochemistry to determine the interactions of a Tat peptide, Tat(38-72), with the loop region of TAR RNA under physiological conditions. A TAR RNA construct with a single 4-thioU residue at positions U31 in the loop sequence was synthesized by chemical methods. Upon UV irradiation, 4-thioU at U31 formed a covalent cross-link with the Tat peptide. We did not observe any RNA-RNA cross-link formation. Competition experiments revealed that a specific RNA-protein complex formation was necessary for the RNA-protein cross-linking reaction. Our results demonstrate that, during RNA-protein recognition, the Tat peptide is located in close proximity to O4 of U31 in the TAR RNA loop sequence.