UV Thermal Melting Curves of tRNA[superscript Phe] in the Presence of Ligands

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which the thermal unfolding of tRNA[superscript Phe] is studied with UV-vis spectrophotometry. Over the course of two laboratory periods, students examine the effects of magnesium and small molecule ligands on tRNA[superscript Phe] conformational stability. The data collected in this laboratory are fit to saturation binding curves, the ligand-binding equilibrium constants are determined, and conclusions are drawn about tRNA[superscript Phe] ligand binding modes and the impact of various types of ligands on tRNA structure. (Contains 1 table.)