Limits...
DNA mimicry by a high-affinity anti-NF-kappaB RNA aptamer.

Reiter NJ, Maher LJ, Butcher SE - Nucleic Acids Res. (2007)

Bottom Line: The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking.Thus, complex formation involves both pre-formed and induced fit binding interactions.The high affinity of the NF-kappaB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Rochester, MN, USA.

ABSTRACT
The binding of RNA molecules to proteins or other ligands can require extensive RNA folding to create an induced fit. Understanding the generality of this principle involves comparing structures of RNA before and after complex formation. Here we report the NMR solution structure of a 29-nt RNA aptamer whose crystal structure had previously been determined in complex with its transcription factor target, the p50(2) form of NF-kappaB. The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking. Remarkably, the free RNA aptamer structure possesses a major groove that more closely resembles B-form DNA than RNA. Upon protein binding, changes in RNA structure include the kinking of the internal loop and distortion of the terminal tetraloop. Thus, complex formation involves both pre-formed and induced fit binding interactions. The high affinity of the NF-kappaB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA.

Show MeSH

Related in: MedlinePlus

Global structure of the free and bound anti-NF-κB RNA conformations. (A) Stereo view of the superimposition over all non-hydrogen atoms of the 10 lowest energy solution structures (RMSD ∼0.94 Å). The 5′ (nt 1–13) and 3′ (nt 18–29) termini of the RNA are colored cyan and blue, respectively, while the GUAA tetraloop is colored gray. (B) Stereo view of the superimposition over just the lower helix. The NMR structure is colored according to Figure 3A and the crystal structure is shown in red (nt 1–17) and pink (nt 18–29).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275087&req=5

Figure 3: Global structure of the free and bound anti-NF-κB RNA conformations. (A) Stereo view of the superimposition over all non-hydrogen atoms of the 10 lowest energy solution structures (RMSD ∼0.94 Å). The 5′ (nt 1–13) and 3′ (nt 18–29) termini of the RNA are colored cyan and blue, respectively, while the GUAA tetraloop is colored gray. (B) Stereo view of the superimposition over just the lower helix. The NMR structure is colored according to Figure 3A and the crystal structure is shown in red (nt 1–17) and pink (nt 18–29).

Mentions: Structures of the anti-NF-κB RNA aptamer were calculated with 541 NOE-derived distance restraints, 160 backbone torsion angle restraints for the helical regions, 25 hydrogen bond restraints based on NOESY and 2JNN COSY analysis, and 19 residual dipolar couplings incorporated at the refinement stage (Figure 2 and Table 1). The structural statistics and stereo superimposition of the 10 lowest-energy structures are shown (Table 1 and Figure 3A). The lower (nt 1–5, 25–29) and upper (nt 10–13, 18–21) helical regions adopt predominantly A-form geometries, and the GUAA fold has the same stacking and hydrogen bonding patterns as previously observed for GNRA tetraloop structures, despite the presence of an adjacent U13-G18 wobble pair (Figures 3 and 4A) (36,37). In contrast, the internal loop deviates from a typical A-form fold (Figures 3B and 4B). Unpaired pyrimidines 6 and 7 contain aromatic stacking interactions within the helix while C24 is stacked between the two uridines. Cross-strand stacking of G8 and G23 reduces the interphosphate cross-strand distance by 3 Å, down to 15 Å in comparison to a regular A-form strand width of 18.5 Å. The non-A-form helical geometry of the internal loop structure appears to be largely due to this cross-strand stack. Cross-strand stacking interactions involving G-A pairs are relatively common in RNA structures (38–41).Figure 3.


DNA mimicry by a high-affinity anti-NF-kappaB RNA aptamer.

Reiter NJ, Maher LJ, Butcher SE - Nucleic Acids Res. (2007)

Global structure of the free and bound anti-NF-κB RNA conformations. (A) Stereo view of the superimposition over all non-hydrogen atoms of the 10 lowest energy solution structures (RMSD ∼0.94 Å). The 5′ (nt 1–13) and 3′ (nt 18–29) termini of the RNA are colored cyan and blue, respectively, while the GUAA tetraloop is colored gray. (B) Stereo view of the superimposition over just the lower helix. The NMR structure is colored according to Figure 3A and the crystal structure is shown in red (nt 1–17) and pink (nt 18–29).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275087&req=5

Figure 3: Global structure of the free and bound anti-NF-κB RNA conformations. (A) Stereo view of the superimposition over all non-hydrogen atoms of the 10 lowest energy solution structures (RMSD ∼0.94 Å). The 5′ (nt 1–13) and 3′ (nt 18–29) termini of the RNA are colored cyan and blue, respectively, while the GUAA tetraloop is colored gray. (B) Stereo view of the superimposition over just the lower helix. The NMR structure is colored according to Figure 3A and the crystal structure is shown in red (nt 1–17) and pink (nt 18–29).
Mentions: Structures of the anti-NF-κB RNA aptamer were calculated with 541 NOE-derived distance restraints, 160 backbone torsion angle restraints for the helical regions, 25 hydrogen bond restraints based on NOESY and 2JNN COSY analysis, and 19 residual dipolar couplings incorporated at the refinement stage (Figure 2 and Table 1). The structural statistics and stereo superimposition of the 10 lowest-energy structures are shown (Table 1 and Figure 3A). The lower (nt 1–5, 25–29) and upper (nt 10–13, 18–21) helical regions adopt predominantly A-form geometries, and the GUAA fold has the same stacking and hydrogen bonding patterns as previously observed for GNRA tetraloop structures, despite the presence of an adjacent U13-G18 wobble pair (Figures 3 and 4A) (36,37). In contrast, the internal loop deviates from a typical A-form fold (Figures 3B and 4B). Unpaired pyrimidines 6 and 7 contain aromatic stacking interactions within the helix while C24 is stacked between the two uridines. Cross-strand stacking of G8 and G23 reduces the interphosphate cross-strand distance by 3 Å, down to 15 Å in comparison to a regular A-form strand width of 18.5 Å. The non-A-form helical geometry of the internal loop structure appears to be largely due to this cross-strand stack. Cross-strand stacking interactions involving G-A pairs are relatively common in RNA structures (38–41).Figure 3.

Bottom Line: The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking.Thus, complex formation involves both pre-formed and induced fit binding interactions.The high affinity of the NF-kappaB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Rochester, MN, USA.

ABSTRACT
The binding of RNA molecules to proteins or other ligands can require extensive RNA folding to create an induced fit. Understanding the generality of this principle involves comparing structures of RNA before and after complex formation. Here we report the NMR solution structure of a 29-nt RNA aptamer whose crystal structure had previously been determined in complex with its transcription factor target, the p50(2) form of NF-kappaB. The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking. Remarkably, the free RNA aptamer structure possesses a major groove that more closely resembles B-form DNA than RNA. Upon protein binding, changes in RNA structure include the kinking of the internal loop and distortion of the terminal tetraloop. Thus, complex formation involves both pre-formed and induced fit binding interactions. The high affinity of the NF-kappaB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA.

Show MeSH
Related in: MedlinePlus