Altering the intermediate in the equilibrium folding of unmodified yeast tRNA(Phe) with monovalent and divalent cations

The isothermal equilibrium folding of the unmodified yeast tRNA(Phe) is stu died as a function of Na+, Mg2+, and urea concentration with hydroxyl radic al protection, circular dichroism, and diethyl pyrocarbonate (DEPC) modific ation. These assays indicate that this tRNA folds in Na+ alone. Similar to folding in Mg2+, folding in Na+ can be described by two transitions, unfold ed-to-intermediate-to-native. The I-to-N transition has a Na+ midpoint of s imilar to0.5 M and a Hill constant of similar to4. Unexpectedly, the urea m -value, the dependence of free energy on urea concentration, for the I-to-N transition is significantly smaller in Na+ than in Mg2+, 0.4 versus 1.7 kc al mol(-1) M-1, indicating that more structure is formed in the Mg2+-induce d transition. DEPC modification indicates that the I state in Na+-induced f olding contains all four helices of tRNA and the I-to-N transition primaril y corresponds to the formation of the tertiary structure. In contrast, the intermediate in Mg2+-induced folding contains only three helices, and the I -to-N transition corresponds to the formation of the acceptor stem plus ter tiary structure. The cation dependence of the intermediates arises from the differences in the stability of the acceptor stem and the tertiary structu re. The acceptor stem is stable at a lower Na+ concentration than required for the tertiary structure formation. The relative stability is reversed in Mg2+ so that the acceptor stem and the tertiary structure form simultaneou sly in the I-to-N transition. These results demonstrate that formation of t he RNA secondary structure can be independent or coupled to the formation o f the tertiary structure depending on their relative stability in monovalen t and divalent ions.