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.
The kink‐turn is a helix‐internal loop‐helix with a three‐nucleotide bulge loop containing a canonical stem (Watson‐Crick base pairing) on one side and non‐canonical stem containing two or three G•A sheared base pairs on the other side. The sheared base pairs in the non‐canonical stem give the construct the flexibility to adopt a 120° kink between the two stems. The kink‐turn is expected to form in the presence of metal‐ions to neutralize the charges on phosphates that are in close proximity. RNA constructs were designed to mimic the Haloarcula Marismortui KT7 from the 28S ribosome. Thermodynamic properties of constructs were tested in a 1 M KCl salt buffer and a 10 mM magnesium buffer at either pH 5.5 or 7. The wild‐type construct shows a −2.36 kcal/mol gain in stability in the magnesium buffer compared to the 1 M KCl buffer at pH 7. Direct measurements of the heat released upon ion‐binding to RNA is being measured using ITC to determine the enthalpy and binding constants of RNA‐magnesium interactions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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