Acquisition of native conformation of ribosomal 5S RNA from E. coli. Hydrodynamic and spectroscopic studies on the unfolding and refolding of the RNA

Abstract

In a continuing effort to decipher the molecular mechanism of ribosome self-assembly [e.g., Dunn, J. M., & Wong, K.-P. (1979) Biochemistry 18, 4380-4385], the mechanism of folding of 5S RNA was investigated by unfolding and refolding studies using several physical techniques including circular dichroism (CD), UV absorption spectroscopy, and sedimentation velocity analysis to monitor various conformational changes. The 5S RNA was unfolded by using 6 M urea and EDTA, and an unfolded state was characterized in which the base pairing was found to be disrupted, but extensive base stacking remained. The unfolded 5S RNA was then refolded upon removal of urea and EDTA by dialysis against a reconstitution buffer both with and without Mg2+, and the refolded states were characterized. The results indicate that under the proper conditions, 5S RNA refolds to a conformation and overall shape very similar to the native conformation. These results indicate that the nucleotide sequence in 5S RNA contains the necessary information to direct the folding of the RNA into its native conformation. The presence of an appropriate concentration of Mg2+ and an incubation at 60 degrees C are required for the correct refolding, since omission of either one results in a renatured 5S RNA whose conformation is quite different from the native one.