Visualization of ion-dependent conformational changes in Escherichia coli 23 S rRNA by scanning transmission electron microscopy

Abstract Electron micrographs of Escherichia coli 23 S rRNA molecules obtained by scanning transmission electron microscopy, unstained and under nondenaturing conditions, reveal previously unresolved structural patterns. The complexity of the pattern is dependent upon the ambient ionic strength conditions. In water and in very low ionic strength buffer, the conformation of 23 S rRNA is characterized by an extended framework, with short side branches related to the secondary and tertiary structure of the molecule. The total length of this filamentous complex is ∼2500 A, only about one-fourth of the length of 23 S rRNA when fully stretched under the denaturing conditions used for imaging by conventional electron microscopy. These data, supplemented by the determination of the linear density ( M L ), suggest that in low ionic strength the backbone of 23 S rRNA is formed by a structure corresponding, on the average, to the mass of four nucleotide strands ( M L ≅ 480 Da A ). With increasing ionic strength, 23 S rRNA coils into more compact forms. Molecules in these states can be characterized by apparent radii of gyration ( R G ), which can be calculated from the mass distribution within the digitized images of individual RNA molecules. The 23 S rRNA is in its most condensed form ( R G = 115 A ) in ribosomal reconstitution buffer; however, it still does not attain the compactness of the large subunit ( R G = 69 A ), nor does it show any resemblance to the native 50 S subunit. The net content of ordered secondary structure, as determined by circular dichroism spectroscopy, is not visibly affected by the changes of ionic strength conditions. These results imply that the observed conformational changes in 23 S rRNA are caused by intramolecular folding of the 23 S rRNA strands induced by the shielding effect of ambient charges.