Thermodynamic Examination of P3a Bulge Loop of a Glycine Riboswitch

Abstract

The glycine riboswitch (GRS) is a non-coding RNA which regulates expression of glycine cleavage and transport genes through transcription attenuation. The riboswitch contains a tandem aptamer which binds glycine to cause structural changes in the downstream expression platform. The 4×3-nt P3a bulge loop motif is implicated in glycine binding in both aptamers. Glycine hydrogen bonds to U78 in the bulge loop along with at least one magnesium ion in the crystal structure. We designed RNA constructs containing a 4×3-nt bulge loop to measure thermodynamics of the P3a bulge loop motif seen in aptamer 1 of V. cholerae. Our goalwasto examine if the bulge loop would bind to glycine in the absence of the larger RNA. We measured the binding of glycine using isothermal calorimetry (ITC) in 10 mM MgCl2 and 1 M KCl buffers at pH 7. A U78A modified construct was also analyzed. Although both structures formed in solution, the wild-type construct did not bind glycine, suggesting that the bulge loop motif does not independently support glycine binding within the riboswitch. Initial data analysis on hybridization of the wild-type RNA structure showed an increase in stability of ~3 kcal/mol in the presence of 10 mM MgCl2 over 1 M KCl. The thermodynamics of the U78A construct was not affected by the presence of magnesium, with only a 0.03 kcal/mol increase in stability in 10 mM MgCl2 buffer.