tRNA conformation and magnesium binding. A study of a yeast phenylalanine-specific tRNA by a fluorescent indicator and differential melting curves.
The binding of Mg2+ to tRNAPhe (yeast) in three conformational states was studied at 10, 30, 45, and 70 degrees 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 were well fitted by two classes of noninteracting binding sites with stability constants independent of temperature (KA = 9X10(4), KB = 6X10(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 degrees C (KB = 3.3X103 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 Mg2+ on the stability of the conformational elements of tRNAPhe (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 Mg2+, whereas all other melting transitions are only influenced by Mg2+ 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 Mg2+ with the 5 strong sites in the folded conformation of tRNA can be ruled out. Strong binding of Mg2+ to completely folded tRNA does not produce a conformational transition changing ultraviolet absorbance, circular dichroism and sedimentation coefficient.
D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium; D008433:Mathematics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010649:Phenylalanine; D012343:RNA, Transfer; D012441:Saccharomyces cerevisiae; D013050:Spectrometry, Fluorescence; D013696:Temperature
D005456:Fluorescent Dyes; D010649:Phenylalanine; D012343:RNA, Transfer; D008274:Magnesium