Equilibrium and kinetics of thermal unfolding of yeast 5.8S ribosomal RNA in aqueous salt solutions

Abstract

Equilibrium and kinetics of thermal melting of yeast 5.8S ribosomal RNA in aqueous NaCl were investigated by differential thermal melting and temperature jump methods. Two peaks were observed in each of the melting curves at 1 mM-1 M Na + and linearity between each melting temperature T m and log[Na +] was found at [Na +> 10 mM. From the difference spectrum ratio, dA 280 dA 260 , the G-C content in the local structures was calculated to be 91 and 56%. The temperature jump to 70–85°C in aqueous 30 mM Na + of the RNA solution induced first-order kinetics, from which the kinetically determined melting curve was calculated. The curve could be approximately described in a Gaussian form with a T m which agrees well with the high T m in the static melting curve at 30 mM Na +. The kinetic properties of the reaction indicated a double helix-coil transition. However, the temperature jump to 20–60°C did not induce monophasic kinetics. The kinetic amplitude of the slow component showed a T m which corresponded to the low T m in the static melting curve at 30 mM Na +. The slow relaxation had the characteristics of a double helix-to-coil transition. However, contributions from very fast processes including single strand unstacking, were most noticeable in the low temperature melting region of the static curve. The thermodynamic parameters of both transitions from double helix to coil were analysed in detail. Both activation energies for helix formation were negative, and the nucleation is thought to follow a process similar to that in oligonucleotides. Values of T m and enthalpy change of both helix-coil transitions indicated the cloverleaf model as the most plausible one for some limited regions of yeast 5.8S RNA among the previously proposed models: burp gun, cloverleaf and Rubin's models.