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Exploring TAR-RNA aptamer loop-loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance.

Lebars I, Legrand P, Aimé A, Pinaud N, Fribourg S, Di Primo C - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: CNRS-Université Bordeaux 1-ENITAB, UMR 5248 CBMN, Institut Européen de Chimie et Biologie, Pessac, France.

ABSTRACT
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.

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Related in: MedlinePlus

Hydrogen bonding in TAR–R06 and TAR–TAR*(GA) kissing complexes. (A) TAR C5 and C6 interactions with R06 U6* and G5* are displayed. The Fo–Fc electron density map is contoured at 1 sigma. Hydrogen bonds and distances are displayed. Distances consistent with the formation of hydrogen bonds are shown. Possible hydrogen bonds are indicated by dotted lines. (B and C) Similar to A for TAR C5 and C6 interaction with TAR*(GA) U6* and G5* for the liquid-crystal NMR (NMR) and the Molecular Dynamics (modelling) structures.
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Figure 3: Hydrogen bonding in TAR–R06 and TAR–TAR*(GA) kissing complexes. (A) TAR C5 and C6 interactions with R06 U6* and G5* are displayed. The Fo–Fc electron density map is contoured at 1 sigma. Hydrogen bonds and distances are displayed. Distances consistent with the formation of hydrogen bonds are shown. Possible hydrogen bonds are indicated by dotted lines. (B and C) Similar to A for TAR C5 and C6 interaction with TAR*(GA) U6* and G5* for the liquid-crystal NMR (NMR) and the Molecular Dynamics (modelling) structures.

Mentions: The presence of additional inter-backbone interactions has been previously proposed and detected in the MD and NMR studies (28,29). In the crystal structure four measured inter-atomic distances are consistent with the establishment of hydrogen bonds and could contribute to non-canonical RNA–RNA interactions (Figure 3A). The G5* (H-O2′) is able to form hydrogen bonds either with C6(O2p) or C5(03′) (distances of 2.57 Å and 3.38 Å, respectively, Supplementary Table 1). The C5(H-O2′) can form hydrogen bonds with U6* (O1p) or U6*(O2p) (distances of 3.33 Å and 2.79 Å, respectively). This hydrogen bonds network differs in the liquid-crystal NMR structure (Figure 3B, Supplementary Table 1) and only one distance is consistent with the formation of a hydrogen bond in the simulated structure (Figure 3C, Supplementary Table 1).Figure 3.


Exploring TAR-RNA aptamer loop-loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance.

Lebars I, Legrand P, Aimé A, Pinaud N, Fribourg S, Di Primo C - Nucleic Acids Res. (2008)

Hydrogen bonding in TAR–R06 and TAR–TAR*(GA) kissing complexes. (A) TAR C5 and C6 interactions with R06 U6* and G5* are displayed. The Fo–Fc electron density map is contoured at 1 sigma. Hydrogen bonds and distances are displayed. Distances consistent with the formation of hydrogen bonds are shown. Possible hydrogen bonds are indicated by dotted lines. (B and C) Similar to A for TAR C5 and C6 interaction with TAR*(GA) U6* and G5* for the liquid-crystal NMR (NMR) and the Molecular Dynamics (modelling) structures.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602780&req=5

Figure 3: Hydrogen bonding in TAR–R06 and TAR–TAR*(GA) kissing complexes. (A) TAR C5 and C6 interactions with R06 U6* and G5* are displayed. The Fo–Fc electron density map is contoured at 1 sigma. Hydrogen bonds and distances are displayed. Distances consistent with the formation of hydrogen bonds are shown. Possible hydrogen bonds are indicated by dotted lines. (B and C) Similar to A for TAR C5 and C6 interaction with TAR*(GA) U6* and G5* for the liquid-crystal NMR (NMR) and the Molecular Dynamics (modelling) structures.
Mentions: The presence of additional inter-backbone interactions has been previously proposed and detected in the MD and NMR studies (28,29). In the crystal structure four measured inter-atomic distances are consistent with the establishment of hydrogen bonds and could contribute to non-canonical RNA–RNA interactions (Figure 3A). The G5* (H-O2′) is able to form hydrogen bonds either with C6(O2p) or C5(03′) (distances of 2.57 Å and 3.38 Å, respectively, Supplementary Table 1). The C5(H-O2′) can form hydrogen bonds with U6* (O1p) or U6*(O2p) (distances of 3.33 Å and 2.79 Å, respectively). This hydrogen bonds network differs in the liquid-crystal NMR structure (Figure 3B, Supplementary Table 1) and only one distance is consistent with the formation of a hydrogen bond in the simulated structure (Figure 3C, Supplementary Table 1).Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: CNRS-Université Bordeaux 1-ENITAB, UMR 5248 CBMN, Institut Européen de Chimie et Biologie, Pessac, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus