Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop
- Allison A. O'Connell,
- Jared A. Hanson,
- Darryl C. McCaskill,
- Ethan T. Moore,
- Daniel C. Lewis and
- Neena Grover
- Corresponding author: ngrover{at}coloradocollege.edu
Abstract
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 C67•A79 base pair that forms an additional hydrogen bond upon protonation, C67•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.
Keywords
- RNA 1 × 2 loop
- single-nucleotide bulge loop
- magnesium ion–RNA interactions
- RNA thermodynamics
- internal loops
- spliceosomal RNA
Footnotes
-
Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.070466.119.
-
Freely available online through the RNA Open Access option.
- Received January 25, 2019.
- Accepted September 19, 2019.
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.