The hydrodynamic and spectroscopic properties of 16 S RNA from Escherichia coli ribosome in reconstitution buffer.

The hydrodynamic shape, conformation, and thermal stability of ribosomal16 S RNA in reconstitution buffer have been studied by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results are compared with the corresponding properties of the 30 S ribosomal subunit in order to understand the role of the RNA molecule in the assembly of the 30 S subunit. Phenolextracted 16 S RNA in reconstitution buffer was found to have a sedimentation coefficient, s&,~ = 21.1 S, a frictional coefficient ratio, f/fnlin = 2.15, and an intrinsic viscosity, [s] = 14.3 ml/g. These results are significantly different from those obtained in other buffers. The corresponding effective hydrodynamic radius is approximately 115 A which is significantly lower than results previously reported. Similar studies were conducted on 16 S RNA prepared by the acetic acid/urea method of Craven and co-workers. The 16 S RNA prepared by this method was reported to have six or seven new protein-binding sites. No differences in conformation and hydrodynamic shape were detected between the two 16 S RNA preparations and only a small difference in thermal stability was observed. A comparison of these results with those obtained for the 30 S subunit conducted under identical conditions indicates that the effective hydrodynamic radii of the 30 S subunit and the 16 S RNA are similar. Furthermore, CD studies indicate only subtle differences between the conformation of 16 S RNA free in solution and that within the 30 S subunit. Melting studies indicate that the proteins stabilize the RNA since the denaturation profile is less cooperative for the 16 S RNA and the melting temperature for the 16 S RNA is lower than that for the 30 S subunit. These results suggest that the 16 S RNA free in solution is already folded into a unique three-dimensional structure before the ribosomal proteins bind to it to assemble into a functional 30 S subunit.