Biomedical subjects

Peter B Rupert

Publications and source records attributed to Peter B Rupert.

3 recordsLinked to original sources

PubMed2004

Crystallization of the hairpin ribozyme: illustrative protocols.

Conditions and techniques that result in successful crystallization differ from RNA to RNA. However, there are some general principles that facilitate crystallization of most RNAs. Three procedures that were instrumental in obtaining well-ordered crystals of the hairpin ribozyme are described in this chapter. These are: i) the design of a series of candidate crystallization constructs; ii) the evaluation of conditions to obtain monodisperse RNA; and iii) the use of seeding techniques to separate nucleation and growth events during crystallization. These procedures can be usefully adapted for the crystallization of other RNAs.

Binding Sites↗

PubMed2003

U1A RNA-binding domain at 1.8 A resolution.

The human U1A RNA-binding domain (RBD1) adopts one of the most common protein folds, the RNA-recognition motif, and is a paradigm for understanding RNA-protein interactions. A 2.8 A resolution structure of the unbound RBD1 has previously been determined [Nagai et al. (1990). Nature (London), 348, 515-520] and revealed a well defined alpha/beta core with disordered termini. Using a longer construct, a 1.8 A resolution structure of the unbound domain was determined that reveals an ordered C-terminal helix. The presence of this helix is consistent with a solution structure of the free domain [Avis et al. (1996). J. Mol. Biol. 257, 398-411]; however, in the solution structure the helix occludes the RNA-binding surface. In the present structure, the helix occupies a position similar to that seen in a 1.9 A resolution RNA-RBD1 complex structure [Oubridge et al. (1994). Nature (London), 372, 432-438]. The crystals in this study were grown from 2.2 M sodium malonate. It is possible that the high salt concentration helps to orient the C-terminal helix in the RNA-bound conformation by strengthening hydrophobic interactions between the buried face of the helix and the alpha/beta core of the protein. Alternatively, the malonate (several molecules of which are bound in the vicinity of the RNA-binding surface) may mimic RNA.

Amino Acid Motifs↗

PubMed2002

Transition state stabilization by a catalytic RNA.

The hairpin ribozyme catalyzes sequence-specific cleavage of RNA through transesterification of the scissile phosphate. Vanadate has previously been used as a transition state mimic of protein enzymes that catalyze the same reaction. Comparison of the 2.2 angstrom resolution structure of a vanadate-hairpin ribozyme complex with structures of precursor and product complexes reveals a rigid active site that makes more hydrogen bonds to the transition state than to the precursor or product. Because of the paucity of RNA functional groups capable of general acid-base or electrostatic catalysis, transition state stabilization is likely to be an important catalytic strategy for ribozymes.

Binding Sites↗