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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 transcripts. We have used an intramolecular RNA self-cleaving strategy to determine the folding of TAR RNA and its interactions with a Tat peptide. We incor-porated 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, RNA self-cleavage was initiated by the addition of an iron salt, ascorbate and hydrogen peroxide. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Sites of RNA cleavage were 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-TAR complex. Our high-resolution footprinting results suggest that the inhibition 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 EDTA-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 peptide. Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures and RNA-protein interactions.

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