Quantitative structural analysis of eukaryotic ribosomal RNA by scanning transmission electron microscopy.

Abstract

The conformation of 28S ribosomal RNA isolated from baby hamster kidney cells was studied by scanning transmission electron microscopy (STEM) and circular dichroic spectroscopy to establish the conditions under which STEM images of unstained freeze-dried rRNA are a meaningful representation of the conformation of rRNA in solution. We have determined the conformation of 28S rRNA under various buffer conditions, the molecular mass, the mass distribution, and the number of polynucleotide strands within the individual molecules, and the apparent radii of gyration. The 28S rRNA molecule is highly extended in water and becomes compact with increasing ionic strength. However, even in the "reconstitution buffer" (30 mM Tris/HCI/20 mM MgCI2/360 mM KCI, pH 7.6) the compactness does not reach a state in which the rRNA molecule appears structurally similar to the 60S ribosomal subunit. Our approach has a broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.It generally has been believed that the conformation of rRNAs in situ and in solution, under certain conditions, is similar and mainly responsible for the shape of the Escherichia coli ribosomal subunits (see ref. 1 for review). However, the picture that emerges from recent studies on the conformation of prokaryotic rRNAs in solution (2-5) is that isolated 16S and 23S RNAs, even in the "reconstitution" buffer, have a more extended conformation than in the ribosome.Eukaryotic ribosomes, which consist of 70-85 different proteins and four RNAs (5S, 5.8S, 18S, and 28S), have been studied to a much lesser extent than their prokaryotic counterparts (6). The primary structure of a limited number of eukaryotic rRNAs has been determined for a variety of species (7-21). Tentative models for the secondary structure of eukaryotic rRNA (10,16,17,19,22), with "domains" similar to those proposed for rRNAs from E. coli (23)(24)(25)(26), have been based on identification of base-paired or chemically crosslinked fragments, chemical modification or enzymatic digestion of single-stranded regions, and comparative sequence analysis. At present, the most direct technique to study the structure ofindividual biological macromolecules is scanning transmission electron microscopy (STEM). The high contrast in dark field and the high efficiency in detecting scattered electrons in STEM make it possible to visualize freeze-dried specimens at high resolution with a low radiation dose and without the need for staining o aiietal shadowing (4,27). One can determine the total mass of biological macromolecules as well as the mass distribution within their structural domains.We have used dedicated STEM to visualize the conformation of isolated 28S rRNA under various buffer conditions, map the mass distribution within the molecule, and determine the apparent radius of gyration (RG). CD spectroscopy was used to monitor changes in the secondary structure of RNA. MATERIALS AND METHODSCell Culture aild RNA Isolation. Baby hamster kidney (BHK) cells ...

Visualization of conformational variability in the domains of long single-stranded RNA molecules

“…We observed individual domain structures corresponding to five of the canonical domains: the 3′ region of canonical domain I, the 3′ region of canonical domain II, most of canonical domain IV, the 5′ region of canonical domain V, and most of canonical domain VI. Overall, the structural detail we have been able to extract using this method seems to exceed that of previous electron microscopy studies ( 25 , 26 ). We were also able to observe flexible regions of the molecule which are difficult to observe in the 3D structures.…”

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy

“…were carried out as in [19]. The apparent values of radii of gyration (RG) were calculated from the distribution of the measured intensities of scattered electrons within the individual RNA molecules [20]. The average number of molecules measured in each experiment was between 100 and 200.…”