Nucleotides in 16S rRNA protected by the association of 30S and 50S ribosomal subunits

We have studied the interaction of 16S rRNA in 30S subunits with 50S subunits using a series of chemical probes that monitor the accessibility of the RNA bases and backbone. The probes include 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT; to probe U at N-3 and G at N-1), diethylpyrocarbonate (DEPC; to probe A at N-7), dimethyl sulfate (DMS; to probe A at N-1, and C at N-3), kethoxal (to probe G at N-1 and N-2), hydroxyl radicals generated by free Fe(II)-EDTA (to probe the backbone ribose groups) and Pb(II). The sites of reaction were identified by primer extension of the probed RNA. Association of the subunits protects the bases of 11 nucleotides and the ribose groups of over 90 nucleotides of 16S rRNA. The nucleotides protected from the base-specific probes are often adjacent to one another and surrounded by sugar-phosphate backbone protections; thus, the results obtained with the different probes confirmed each other. Most of the protected nucleotides occur in five extended-stem-loop structures around positions 250, 700, 790, 900, and 1408-1495. These regions are located in the platform and bottom of the subunit in the general vicinity of inter-subunit bridges that are visible in reconstructed electron micrographs. Our results provide an extensive map of the nucleotides in 16S rRNA that are likely to be involved in subunit-subunit interactions.