Role of divalent ions in folding of tRNA.
The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 degrees C), but can be restored by addition of divalent ions. We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4-thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the 'Y' base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance. The fluorescence of the reduced 8-13 photoproduct (in which 4-thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed. In low salt (e.g. 4.5 mM Na+), the region of 4-thiouracil is affected strongly as the first eight Mn2+ bind to tRNA, whereas the fluorescence of the 'Y' base is affected only after four Mn2+ are bound. Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later. The binding of divalent ions to each region starts together with its restructuration. Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+. If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region. Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure. These observations suggest that the  role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese-phosphate binding supplemented by electrostatic interaction with distant phosphates.
D000926:Anticodon; D001665:Binding Sites; D002413:Cations, Divalent; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium; D008345:Manganese; D009239:N-Formylmethionine; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer; D012263:Ribonucleosides; D012964:Sodium; D013889:Thiouracil
D000926:Anticodon; D002413:Cations, Divalent; D012263:Ribonucleosides; D009239:N-Formylmethionine; D008345:Manganese; D013889:Thiouracil; D012343:RNA, Transfer; D012964:Sodium; D008274:Magnesium