Thermodynamic examination of U6 ISL bulged RNA

Abstract

A highly conserved bulge in the internal stem‐loop (ISL) of the U6 subunit of the spliceosome is responsible for its assembly and catalysis. This pH dependent bulge has an unpaired U80 nucleotide stacked within the helix that causes a bend in the RNA at pH 7.0. At a lower pH the flanking A•C base pair becomes protonated (forming an A+•C base pair) causing U80 to be flipped out into the major groove allowing the RNA to take up a conformation close to A‐form helix. We tested constructs containing the conserved bulged nucleotides as well as those with modifications in both the bulged and flanking helical nucleotides in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. Only constructs containing the A•C pair showed a gain in stability at pH 5.5 over 7.5. All constructs, with one exception, showed an increase in stability with increase in Mg2+ concentrations. RNA constructs also show greater gain in stability in the presence of Mg2+ than DNA constructs. Our results confirm that lowering the pH allows for the formation of an A+•C base pair in 1 M KCl and in 0.5 to 9.5 mM Mg2+ giving an extra stability of more than 1 kcal/mol in the wild type construct. This work was funded by NSF Grant MCB‐ 0950582 to NG.