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We have designed a new guided-inquiry laboratory for an advanced biochemistry course. This integrated laboratory focuses on the biomolecule tRNA[superscript Phe] and combines elements of bioorganic and bioinorganic chemistry with biochemistry. Throughout the semester students work together to study tRNA[superscript Phe] structure and ligand binding by (i) assaying metal-catalyzed hydrolysis using gel electrophoresis; (ii) probing thermal denaturation with UV-vis spectrophotometry; and (iii) characterizing ligand associated changes in conformation via fluorescence spectroscopy. Students begin the course with a common laboratory experience and then diverge into parallel projects, ending with a novel self-designed project. In their final report, students interpret experimental results from the entire class and draw conclusions regarding the effects of various ligands on the structure of tRNA[superscript Phe]. (Contains 1 scheme, 2 tables, 13 figures and 6 notes.)

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 students use fluorescence spectroscopy to study tRNA[superscript Phe] structure. Both intrinsic (Y Base) and extrinsic (ethidium bromide) fluorescence probes are monitored. Ligands (e.g., spermine, neomycin B, magnesium) are titrated into tRNA[superscript Phe] solution and changes in fluorescence intensity are monitored. Students fit the data to hyperbolic saturation curves to determine ligand-binding equilibrium constants and draw conclusions on how different binding modes affect tRNA[superscript Phe] tertiary structure. (Contains 3 tables, 1 figure and 2 notes.)

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 students assay metal-catalyzed hydrolysis of tRNA[superscript Phe] using gel electrophoresis. tRNA[superscript Phe] 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 tRNA[superscript Phe]. Gel bands are quantified to determine ligand-binding equilibrium constants for the small molecule ligand-tRNA[superscript Phe] 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 tRNA[superscript Phe] structure, ligand charge, and inorganic chemistry of the metal ion complexes. (Contains 2 tables, 1 figure and 2 notes.)

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.)

The article focuses on the changes in the conformation of tRNAPhe by using an integrated biochemistry laboratory course. Students use three experimental techniques including gel electrophoresis, UV-vis spectrophotometry, and fluorescence spectroscopy. The class examines the changes in the structure of a ligand that affect its ability to bind and change the tRNAPhe structure. Discussions concerning the projects probing tRNAPhe structure using various assay methods are also presented.

This article presents a study regarding the fluorescence spectroscopy of tRNAPhe Y base in the presence of magnesium2+ and small molecule ligands. It discusses that tRNAPhe contains a unique Y base with three planar aromatic rings and serves as a fluorophore. It serves as an intrinsic probe to study the conformational changes of tRNAPhe in the presence of magnesium ions and small molecule ligands because Y base fluorescence is exquisitely sensitive to the local environment.

This article presents a study regarding the metal-catalyzed cleavage of tRNAPhe. It discusses that Lead(II)-catalyzed hydrolysis is an effective assay of tRNAPhe tertiary structure because it catalyzes the cleavage of native, folded tRNAPhe at a single site in the D loop resulting in a large fragment and a small fragment. In addition, ligand binding alters the conformation of tRNAPhe inhibiting lead-catalyzed cleavage and metals other than lead may also cleave tRNA.

This article presents a study regarding the UV thermal melting curves of tRNAPhe in the presence of ligands. It discusses that native secondary and tertiary structures of tRNAPhe are only stable below about 50-70 degrees celcius because of the positive entropy of unfolding. A laboratory project was designed to investigate the influence of equilibrium ligand binding on the conformational stability of tRNAPhe via UV melting curves. The melting temperature is derived by calculating the derivatives of the absorbance versus temperature curves using the Cary program.