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.

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The bulged‐G motif has been recognized in the crystal structure of ribosomal RNA as it contains an S‐turn in the phosphodiester backbone. The S‐turn is formed by a G•U‐A base triple that has a non‐canonical A‐G base pair above the base triple, and an adenine bound to either a guanine or uracil below the base triple. The base triple contains the bulged G nucleotide and is a binding site for elongation factors which ensure that proper translation occurs; this same RNA also binds to sarcin and ricin that inhibit ribosomal function. A similar S‐shape in an RNA backbone was also found to occur in the bacterial c‐di‐GMP‐II riboswitch that contains a A•C‐G base triple. In this study, thermal denaturation and isothermal titration calorimetry (ITC) experiments were performed to delineate the role of specific nucleotides in the formation of the S‐turn and its interaction with magnesium ions. A wild type RNA construct was derived from the bulged‐G motif that contained the G•U‐A base triple flanked by the non‐canonical base pairs, and an RNA triple substitution construct was designed that replaced the base triple with a A•C‐G. The wild type RNA and the base‐triple‐substituted constructs had similar stabilities in 1 M KCl buffer at pH 7.0 and 5.5. RNA constructs are additionally stabilized in 10 mM magnesium, with 2.63 kcal/mol gain in stability for the wild type and 1.45 kcal/mol for A•G‐C constructs. ITC experiments were performed to measure the enthalpy, entropy, and the binding affinity of magnesium ions to RNA. Results for wild type and modified constructs will be presented.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.