The Conformational Transitions in Yeast tRNAPhe as Studied with tRNAPhe Fragments

Conformational transitions of the anticodon branch, the acceptor branch and the tertiary structure of yeast tRNA Phe were investigated using tRNA Phe fragments. Thermodynamic and kinetic methods were employed using the optical absorbance of the fragments and the fluorescence of the Y‐base. Related results of partial nuclease digestions on tRNA Phe fragments are also reported. Hypochromicity, reaction enthalpy and rate constants of helix‐coil transitions of the anti codon branch agree with the values expected from the cloverleaf model. The dissociation of the branch is accompanied by a quenching of the fluorescence of the Y‐base. In some isolated fragments the anticodon branch is more stable than in the intact tRNA. The destabilization in the intact tRNA is attributed to electrostatic effects. The anticodon half Phe 21–57, at low concentrations and temperatures, assumes a structure which is different from a segment of the cloverleaf model. A transition from this structure to the one of a segment of the cloverleaf is observed with increasing temperature. At higher fragment concentrations and in the presence of Mg 2+ dimers are formed. Bimolecular recombinations are observed between fragments Phe 1–18 and Phe 21–76 reconstituting the acceptor and dihydrouridine stems, and between Phe 1–18 and Phe 38–76 where only the base pairs of the acceptor stem are newly formed. The mechanism of the recombination and the relationship of the fragment combination to the intact tRNA are discussed. The thermodynamic properties of the fragment Phe 1‐69/70 are very similar to those of the intact tRNA. It can be concluded that the tertiary structure is essentially unchanged anti that the terminal nucleotides 70–76 are not directly involved in its formation. The thermal denaturation process of intact tRNA Phe can now be interpreted as follows: at first the tertiary structure is converted to a cloverleaf‐like structure; this process is followed by the melting of the acceptor and anticodon stems; finally the ribosylthymine and dihydrouridine stems dissociate independently.