Binding of metal ions and antibody fragments to folded RNA molecules

The stability and biological functions of folded RNA molecules depend intimately on their interactions with both metal ions and proteins. By screening the negative charges of the phosphate backbone, metal ions provide the necessary ionic environment required for RNAs to fold into complex three-dimensional architectures. Proteins also stabilize RNA structures, often by preventing RNA misfolding or by combining with RNA to form functional ribonucleoprotein particles. Very little RNA exists in living cells that is not bound up with proteins in some way. Thus, the ways in which both metal ions and proteins interact with RNA govern how folded RNAs will perform their biological functions.;This Dissertation presents work on two separate lines of research investigating metal ion- and protein-RNA interactions. The first involves the biochemical detection of site-bound divalent metal ions within folded RNAs. Most metal ions that stabilize folded RNAs exert their effects as part of the diffuse ion atmosphere that surrounds the molecule. However, the electrostatic potential of some regions of the RNA surface is sufficiently negative such that direct, stoichiometric metal ion binding becomes energetically favorable. For the DeltaC209 P4-P6 RNA, phosphorothioate substitution of the oxygen metal ion ligands involved in these interactions can be extremely destabilizing. However, under the right conditions, the addition of a softer metal ion (Mn2+) can rescue a phosphorothioate-induced folding defect. Such metal ion rescue has been used previously to detect metal ion interactions important for ribozyme catalysis. This work shows how a metal ion rescue experiment can be used to detect site-bound metal ions important for equilibrium stabilization of a folded RNA.;The second focus of this Dissertation concerns the interaction of folded RNAs with antibody fragments, or Fabs, selected using phage display methodology. The Fabs were selected to bind specific folded RNAs with the hope that the Fabs would facilitate crystallization of the target RNAs. We have developed a biochemical method of determining the region on the target RNAs where these Fabs bind. Characterization of these RNA binding sites, or epitopes, yields information about the types of RNA structures recognized by proteins in general, and by antibodies in particular.