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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.
Biochemistry 04/1982; 21(9):2096-102. · 3.42 Impact Factor
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ABSTRACT: The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).
Journal of Biological Chemistry 11/1979; 254(20):10139-44. · 4.77 Impact Factor
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ABSTRACT: The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.
Journal of Biological Chemistry 02/1978; 253(1):18-20. · 4.77 Impact Factor
