Metal-Catalyzed Cleavage of tRNAPhe

Abstract

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNAPhe conformational changes induced by ligand binding. In this article we describe a set of experiments in which students assay metal-catalyzed hydrolysis of tRNAPhe using gel electrophoresis. tRNAPhe cleavage patterns are monitored in the presence of lead(II) and other metals cations. Small molecule ligands are also studied to determine their ability to enhance or inhibit lead(II)-catalyzed cleavage of tRNAPhe. Gel bands are quantified to determine ligand-binding equilibrium constants for the small molecule ligand–tRNAPhe adducts. By pooling class data from the metal and small molecule ligand experiments, students draw conclusions regarding how metal-catalyzed hydrolysis is affected by various parameters such as tRNAPhe structure, ligand charge, and inorganic chemistry of the metal ion complexes.