RNA-protein interactions in the ribosome: III. Conformation and stability of ribosomal protein binding sites in the 16 S RNA

Abstract

Following dialysis against distilled water, the 16 S ribosomal RNA of Escherichia coli is unable to interact with 30 S subunit protein S4 at 0 °C. The dialysed RNA recovered this capacity, however, when heated at 40 °C in the presence of 0.02 m-MgCl 2 prior to addition of the protein. Furthermore, its sensitivity to ribo-nuclease markedly declined and its sedimentation rate increased as a consequence of this treatment. Although no concomitant changes in secondary structure were detected by absorbance and fluorescence techniques, the rearrangement of a small number of base-pairs was not excluded. Kinetic measurements revealed that binding site reactivation satisfies the first-order rate law and that the process is highly temperature-dependent, exhibiting an Arrhenius activation energy of 40,800 cal/mol. Together, these data suggest that dialysed RNA undergoes a unimolecular conformational transition upon pre-incubation in Mg 2+-containing buffers and that this transition leads to renaturation of the binding site for protein S4. Similar results were obtained for several other proteins of the 30 S subunit. In particular, S7, S16/S17 and S20 all failed to interact efficiently with dialysed 16 S RNA at 0 °C. These proteins bound normally to the RNA, however, after it had been incubated at 40 °C in the presence of Mg 2+ ions. By contrast, prior dialysis of the 16 S RNA did not affect its ability to associate with S8 and S15 at 0 °C. These two proteins interacted equally well with dialysed and pre-incubated 16 S RNA, indicating that their binding sites are not susceptible to the reversible alterations in conformation which influence the attachment of the other RNA-binding proteins to the nucleic acid molecule. The effects of dialysis and pre-incubation on the interaction of 16 S RNA with an unfractionated mixture of 30 S subunit proteins were also investigated. The dialysed RNA bound only S6, S8, S15 and S18 at 0 °C whereas, after heating at. high Mg 2+ concentrations, the RNA associated with S4, S7, S9, S13, S16/S17, S19 and S20 as well. These results leave little doubt that the protein-binding capacities of the 16 S RNA are intimately related to its three-dimensional configuration, although individual binding sites appear to differ significantly in their stability to small changes in structure.