Thermodynamic Examination of Bulge Sequence, Ion Binding, and Helical Context of 2X2 and 2X1 Bulges in RNA

Abstract

Bulges are non‐helical regions in RNA that provide sites for protein and ligand interactions. The interactions in and around the bulge region are non‐canonical and often not well understood. In crystal structures, bulges and non‐canonical base pairs are often the sites for interactions with metal ions. The pyrophosphate sensor helix of the thiamine pyrophosphate (TPP) riboswitch contains a 2×2 bulge that binds the pyrophosphate group along with Mg2+ ions. The 1×2 internal stem loop of the U6 spliceosomal RNA changes conformation upon the formation of A+•C base pair and Mg2+ binding. In this project, we are investigating the non‐canonical base pairs in the bulge regions, the helical context in which these are found, and the stabilization of these RNA in the presence of Mg2+ ions. Wild type and modified RNA constructs were designed to study RNA stability using thermal denaturation experiments in 1 M KCl and in varying concentrations of Mg2+, in pH 5.5 and 7.5. In 1 M KCl, the wild type U6 2X1 and TPP 2X2 constructs showed a greater stability at pH 5.5 over pH 7.5 likely due to the formation of A+•C base pair. All TPP 2X2 constructs, with one exception, are more stable in 9.5 mM Mg2+ over 1 M KCl. Comparative thermodynamic data will be presented on various constructs to better elucidate the differences and similarities of 2X2 and 1×2 bulge regions. This work was funded by NSF Grant MCB‐0950582 to NG.