Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis of HIV-1 Tat-TAR complex

Replication of human immunodeficiency virus type 1 (HIV-1) requires specifi c 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 tr anscripts. We have used an intramolecular RNA self-cleaving strategy to det ermine the folding of TAR RNA and its interactions with a Tat peptide. We i ncorporated an EDTA analog at position 24 in the HIV-1 Tat binding site of the TAR RNA. After isolation and purification of the EDTA-TAR conjugate, RN A self-cleavage was initiated by the addition of an iron salt, ascorbate an d hydrogen peroxide, Hydroxyl radicals generated from the tethered Fe(II) c leaved TAR RNA backbone in two localized regions. Sites of RNA cleavage wer e mapped by sequencing reactions, A Tat fragment, Tat(38-72), specifically inhibited RNA self-cleavage. To determine the structural changes caused by the Tat peptide, we performed Fe(II)-EDTA footprinting experiments on Tat-T AR complex, Our high-resolution footprinting results suggest that the inhib ition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i ) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage and (ii) RNA goes through a structural change where EDT A-U24 is rigidly positioned out of the helix and cannot get access to other nucleotides in the loop of TAR RNA, which are not protected by the Tat pep tide, Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage c an be applied to study RNA tertiary structures and RNA-protein interactions .