Valsan Mandiyan

Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12.

A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series. This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1. In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions. Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region. This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center. The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head. A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.

[3] Structural analysis of ribosomes by scanning transmission electron microscopy

Publisher Summary This chapter describes that dedicated scanning transmission electron microscopy (STEM) is uniquely suited to high-resolution structural studies on ribosomes and other biological macromolecules. High efficiency in collection of scattered electrons in the STEM dark-field mode makes it possible to visualize unstained freeze-dried ribosomes and their components without the main resolution-limited artifacts of staining and distortion by air-drying and radiation inherent in the conventional transmission electron microscopy (TEM). The linearity of the relationship between scattering cross-section and molecular weight can be utilized for the determination of the molecular mass of ribosomes and their constituents, mass distribution within the particles, and calculation of the apparent radius of gyration. Protein-free deposition of unstained freeze-dried rRNA molecules improved significantly the visualization of their conformation and made it possible to initiate high-resolution studies of RNA-protein interactions and the process of ribosome assembly. Supplementation of STEM with an electron energy loss spectrometer and application of computer image averaging and multivariate statistical analysis of electron micrographs provide additional highly specific and quantitative information on three-dimensional structure, mass, and element distribution in the ribosome for topographical and phylogenetic studies.

Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy

Quantitative mass image analysis of Escherichia coli ribosomal particles by scanning transmission electron microscopy (STEM) provided direct evidence that presumably homogeneous preparations of ribosomes are, in reality, populations of heterogeneous particles. Variations in composition, relative molecular mass (Mr) and shape were observed both in the monosomes and in the ribosomal subunits. None of these changes can be resolved visually; they can be evaluated only by computer processing. The variations in relative mass and shape monitored by values of radius of gyration (RG) were attributed to the loss of ribosomal proteins and/or factors and correlated with the changes in ribosome composition and biological activity. The highest activity was found in monosomes prepared from the standard 0.5 M NH4Cl wash. With increasing concentrations (up to 1.5 M) of NH4Cl in the wash buffer the activity decreased slowly, then dropped rapidly to about half in 2 M NH4Cl. The most striking effects were observed in ribosomal particles washed with 0.1 M NH4Cl. The 70S monosomes and the 30S subunits attained maximum Mr and RG values (2660 kDa and 76 A, and 990 kDa and 75 A, respectively), which were greater than the theoretical values, while the activity was minimal (approximately 12%). The Mr and RG parameters of the 50S subunits remained uneffected by the NH4Cl washes (approximately 1600 kDa and 68 A).

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

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.

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

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 approximately 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 approximately equal to 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 (RG), 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 (RG = 115 A) in ribosomal reconstitution buffer; however, it still does not attain the compactness of the large subunit (RG = 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.