tRNA Conformation and Magnesium Binding: A Study of Yeast Phenylalanine‐Specific tRNA by a Fluorescent Indicator and Differential Melting Curves

Abstract

The binding of Mg 2+ to tRNA Phe (yeast) in three conformational states was studied at 10, 30, 45, and 70°C by the fluorescence indicator 8‐hydroxyquinoline 5‐sulphonic acid in the presence of 0.032 M monovalent cations (Na+). At temperatures below those characteristic for early melting (completely folded tRNA) the Scatchard plots are biphasic. They are well fitted by two classes of noninteracting binding sites with stability constants independent of temperature (KA= 9 × 10 4, KB= 6 × 10 3 M−1). In partially unfolded tRNA the strong binding process is co‐operative. A single class of weak sites was found in the statistically coiled conformation at 70°C (KB= 3.3 × 10 3 M−1). The total number of binding sites is 23 ± 5; differences for the folded and unfolded conformations are smaller than 1. The influence of Mg 2+ on the stability of the conformational elements of tRNA Phe (yeast) and its CCA‐half (i.e. nucleotides 38–76) was determined by differential ultraviolet absorbance and depolarisation melting curves using the fluorescence of the Y base. Tertiary structure corresponding to early melting is stabilized by strongly bound Mg 2+, whereas all other melting transitions are only influenced by Mg 2+ bound at weak sites. The stability constants of tertiary structure obtained from the melting experiments can quantitatively be described by assuming that 5 ± 1 non‐interacting strong sites as characterized by the fluorescence titrations are converted to weak sites upon unfolding of the tertiary structure. Co‐operative interaction of Mg 2+ with the 5 strong sites in the folded conformation of tRNA can be ruled out. Strong binding of Mg 2+ to completely folded tRNA does not produce a conformational transition changing ultraviolet absorbance, circular dichroism and sedimentation coefficient.