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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.

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Anti-NF-κB RNA aptamer and crystal structure in complex with NF-κB p502. (A) Proposed secondary structure of the 29-nt anti-NF-κB RNA aptamer in solution. The RNA hairpin studied here is identical in sequence to the RNA aptamer co-crystallized with NF-κB p502 (with exception of the inversion of terminal nucleotides G1 and C29). Canonical RNA Watson–Crick base pairs are red, the 5′ region of the internal loop is cyan, the wobble pair is green, the GNRA-type tetraloop is gray, and the 3′ region of the internal loop is blue. (B) RNA aptamer structure extracted from the crystal structure and colored as in (A). (C and D) Comparison of the crystal complex of NF-κB p502 with the RNA aptamer (PDB ID 1OOA) or with bound DNA (PDB ID 1NFK). RNA binding requires substantial opening of the Rel homology domains (8,9). The two p50 subunits are shown in orange and yellow. Nucleic acids are shown as space filling models, where the RNA aptamer structures (C) are colored as in (A) and DNA structure (D) is colored red and pink.
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Figure 1: Anti-NF-κB RNA aptamer and crystal structure in complex with NF-κB p502. (A) Proposed secondary structure of the 29-nt anti-NF-κB RNA aptamer in solution. The RNA hairpin studied here is identical in sequence to the RNA aptamer co-crystallized with NF-κB p502 (with exception of the inversion of terminal nucleotides G1 and C29). Canonical RNA Watson–Crick base pairs are red, the 5′ region of the internal loop is cyan, the wobble pair is green, the GNRA-type tetraloop is gray, and the 3′ region of the internal loop is blue. (B) RNA aptamer structure extracted from the crystal structure and colored as in (A). (C and D) Comparison of the crystal complex of NF-κB p502 with the RNA aptamer (PDB ID 1OOA) or with bound DNA (PDB ID 1NFK). RNA binding requires substantial opening of the Rel homology domains (8,9). The two p50 subunits are shown in orange and yellow. Nucleic acids are shown as space filling models, where the RNA aptamer structures (C) are colored as in (A) and DNA structure (D) is colored red and pink.

Mentions: The essential features of the RNA aptamer structure derived from its complex with NF-κB p502 are shown in Figure 1A. The RNA aptamer was predicted to fold as a stem-loop structure with an asymmetric internal loop, and a U–G wobble pair adjacent to a terminal GUAA tetraloop. Examination of the RNA aptamer within the NF-κB-aptamer complex (Figure 1B) validated secondary structure predictions, and revealed structural features deviating from A-form geometry. The latter included a large overall kink in the RNA and a complex pattern of base interactions within the asymmetric internal loop. These interactions included non-canonical pairing of A9–G22 and U6–C24, with stacking of unpaired bases U7, G8 and G23. Together with the U13–G18 wobble pair, these interactions provided a series of hydrogen bonding and van der Waals contacts with the DNA binding surface of NF-κB p50. In the crystal complex with NF-κB p502, one aptamer binds to each NF-κB subunit, requiring a large opening of the Rel homology domains in the protein dimer relative to the structure bound to DNA (Figure 1C and D). In fact, the aptamer structure mimics the major groove of the normal DNA binding sequence in such a manner that the interacting protein side chain conformations are largely preserved between the two complexes (8,10).Figure 1.


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

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

Anti-NF-κB RNA aptamer and crystal structure in complex with NF-κB p502. (A) Proposed secondary structure of the 29-nt anti-NF-κB RNA aptamer in solution. The RNA hairpin studied here is identical in sequence to the RNA aptamer co-crystallized with NF-κB p502 (with exception of the inversion of terminal nucleotides G1 and C29). Canonical RNA Watson–Crick base pairs are red, the 5′ region of the internal loop is cyan, the wobble pair is green, the GNRA-type tetraloop is gray, and the 3′ region of the internal loop is blue. (B) RNA aptamer structure extracted from the crystal structure and colored as in (A). (C and D) Comparison of the crystal complex of NF-κB p502 with the RNA aptamer (PDB ID 1OOA) or with bound DNA (PDB ID 1NFK). RNA binding requires substantial opening of the Rel homology domains (8,9). The two p50 subunits are shown in orange and yellow. Nucleic acids are shown as space filling models, where the RNA aptamer structures (C) are colored as in (A) and DNA structure (D) is colored red and pink.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Anti-NF-κB RNA aptamer and crystal structure in complex with NF-κB p502. (A) Proposed secondary structure of the 29-nt anti-NF-κB RNA aptamer in solution. The RNA hairpin studied here is identical in sequence to the RNA aptamer co-crystallized with NF-κB p502 (with exception of the inversion of terminal nucleotides G1 and C29). Canonical RNA Watson–Crick base pairs are red, the 5′ region of the internal loop is cyan, the wobble pair is green, the GNRA-type tetraloop is gray, and the 3′ region of the internal loop is blue. (B) RNA aptamer structure extracted from the crystal structure and colored as in (A). (C and D) Comparison of the crystal complex of NF-κB p502 with the RNA aptamer (PDB ID 1OOA) or with bound DNA (PDB ID 1NFK). RNA binding requires substantial opening of the Rel homology domains (8,9). The two p50 subunits are shown in orange and yellow. Nucleic acids are shown as space filling models, where the RNA aptamer structures (C) are colored as in (A) and DNA structure (D) is colored red and pink.
Mentions: The essential features of the RNA aptamer structure derived from its complex with NF-κB p502 are shown in Figure 1A. The RNA aptamer was predicted to fold as a stem-loop structure with an asymmetric internal loop, and a U–G wobble pair adjacent to a terminal GUAA tetraloop. Examination of the RNA aptamer within the NF-κB-aptamer complex (Figure 1B) validated secondary structure predictions, and revealed structural features deviating from A-form geometry. The latter included a large overall kink in the RNA and a complex pattern of base interactions within the asymmetric internal loop. These interactions included non-canonical pairing of A9–G22 and U6–C24, with stacking of unpaired bases U7, G8 and G23. Together with the U13–G18 wobble pair, these interactions provided a series of hydrogen bonding and van der Waals contacts with the DNA binding surface of NF-κB p50. In the crystal complex with NF-κB p502, one aptamer binds to each NF-κB subunit, requiring a large opening of the Rel homology domains in the protein dimer relative to the structure bound to DNA (Figure 1C and D). In fact, the aptamer structure mimics the major groove of the normal DNA binding sequence in such a manner that the interacting protein side chain conformations are largely preserved between the two complexes (8,10).Figure 1.

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