U6 RNA contains a 1 × 2-nt internal loop that folds and unfold during spliceosomal assembly and activation. The 1 × 2 loop consists of a C 67 •A 79 base pair that forms an additional hydrogen bond upon protonation, C 67 •A + 79 , and uracil (U80) that coordinates the catalytically essential magnesium ions. We designed a series of RNA and DNA constructs with a 1 × 2 loop sequence contained in the ISL, and its modifications, to measure the thermodynamic effects of protonation and magnesium binding using UV-visible thermal denaturation experiments. We show that the wild-type RNA construct gains 0.43 kcal/mol in 1 M KCl upon lowering the pH from 7.5 to 5.5; the presence of magnesium ions increases its stability by 2.17 kcal/mol at pH 7.5 over 1 M KCl. Modifications of the helix closing base pairs from C-G to U•G causes a loss in protonation-dependent stability and a decrease in stability in the presence of magnesium ions, especially in the C68U construct. A79G single-nucleotide bulge loop construct showed the largest gain in stability in the presence of magnesium ions. The DNA wild-type construct shows a smaller effect on stability upon lowering the pH and in the presence of magnesium ions, highlighting differences in RNA and DNA structures. A U6 RNA 1 × 2 loop sequence is rare in the databases examined.

There is a highly conserved bulge in the internal stem‐loop (ISL) of the U6 subunit of the spliceosome. It is responsible for spliseosome activation and catalysis. The U6 ISL bulge structure is pH dependent. At a pH around 7.0 the unpaired U80 bulged nucleotide is stacked within the helix causing a bend of approximately 25 degrees. At a lower pH U80 is flipped out into the major groove allowing the RNA to take up a conformation close to A‐form helix. There are several single mutations in this bulge that have proven lethal in yeast, and a few single mutations that exacerbate the lethality. Ion binding, pH effects, and the stability of various sequences involving the bulge will be studied using thermodynamics.

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.

Spliceosome is large complex made up of five snRNA and nearly 70 proteins that assembles on pre‐mRNA to excise the introns. The U6 subunit of the spliceosome is required for spliceosome assembly and activation. Spliceosome undergoes number of structural rearrangements that disrupt U4/U6 complex and form U2/U6 complex to become activated. The association of U2/U6 complex results in the formation of a highly conserved internal stem loop (ISL) in U6 RNA. The formation of a 1x2 nucleotide bulge that binds catalytically active Mg2+ at U80 position is essential in the RNA cleavage. The 1x2 bulge contains an A79•C67 non‐canonical base pair, which forms a stronger A+•C base pair in lower pH conditions. Upon the A+•C base pair formation, the ISL adopts a conformation closer to an A‐form helix and the unbound U80 in the bulge is forced out into the major groove. This conformation is mutually antagonistic to the binding of the catalytic Mg2+ ion, and therefore prevents splicing activity. Thermal denaturation experiments were performed on RNA constructs derived to mimic 1x2 bulge of the U6 RNA. We tested constructs containing the conserved bulged nucleotides as well as those with modifications that disrupt spliceosomal function. All constructs were examined in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. The thermodynamic contributions of the bulged RNA were compared to those of the helical RNA and DNA constructs. Thermodynamics of RNA with modification in and around the 1x2 bulge loop will be presented and influence of pH and ionic conditions will be discussed.