Crystallographic and functional studies of a biotin-binding RNA pseudoknot

The structure/function relationship in RNA is studied in the context of the biotin aptamer. The molecule was developed through in vitro selection and binds to the non-aromatic, non-planar carboxylation co-factor biotin. Binding is highly specific with a KD of 6 muM and chemical modification experiments confirm the pseudoknot motif proposed by phylogenetic analysis. Two crystal forms of the molecule have been grown. The tetragonal crystal form diffracts to 2.8 A and is hard to reproduce. The monoclinic crystal form diffracts to 1.3 A and is highly reproducible. The structure of the molecule has been solved to 1.3 A by x-ray crystallography from the monoclinic crystals. The crystal structure represents the highest resolution structure of a pseudoknot and/or aptamer to date and allows the comparison of highly specific binding pockets for the biotin ligand formed by RNA and protein. Six well-defined magnesiums are seen in the structure and display a range of interactions with the RNA with one of the magnesiums directly coordinated both the RNA and the biotin ligand. These interactions are both inner- and outer-sphere and involve both base moieties and backbone atoms. Theoretical calculations of the electrostatic field of the aptamer using the non-linear Poisson-Boltzmann equation coupled with Brownian dynamics simulations of a cation probe were performed to help define the parameters of metal binding in the RNA. Additionally, the crystal structures of the molecule have been solved with the replacement of magnesium with calcium and strontium. The overall fold of the RNA remains unchanged along with certain metal-RNA coordination motifs. However, the metal strontium replaces magnesium at a critical binding site in loop 1 utilizing direct coordination to the N7 of a base in helix 2. Optical temperature melts in increasing amounts of magnesium ion provide the thermodynamic data with which to propose an unfolding model of the aptamer. In this model, the pseudo-continuous helix unstacks with a low enthalpy transition, followed by the melting of helix 1 and helix 2, respectively. This work enhances our understanding of RNA structure at atomic resolution and the general behavior of pseudoknots and their interactions with divalent cations.