Exploring TAR-RNA aptamer loop-loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance.
In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5' untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605-1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson-Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1 degrees , thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2' hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2'-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability.
Lebars
Isabelle
I
CNRS-Université Bordeaux 1-ENITAB, UMR 5248 CBMN, Institut Européen de Chimie et Biologie, Pessac, France.
Legrand
Pierre
P
Aimé
Ahissan
A
Pinaud
Noël
N
Fribourg
Sébastien
S
Di Primo
Carmelo
C
D019380:Anti-HIV Agents / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009691:Nucleic Acid Denaturation; D012367:RNA, Viral / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D020349:Surface Plasmon Resonance
D019380:Anti-HIV Agents; D052157:Aptamers, Nucleotide; D012367:RNA, Viral