Protein-nucleic acid interactions which occur during Escherichia coli 50 S ribosomal subunit assembly between 23 S rRNA, 5 S rRNA and a complete set of 34 L-proteins were monitored by high resolution scanning transmission electron microscopy (STEM). This approach made it possible to visualize and quantitatively analyze conformational changes induced in the rRNAs during E. coli 50 S ribosomal subunit assembly. The reconstituted RNA-protein complexes, the control 23 S rRNA and native 50 S subunits were characterized by their mass and morphology. Association of 23 S rRNA with the first assembly protein, L24, led to the formation of a distinct nucleus of mass ("cluster") on the filamentous and loosely coiled molecule of the 23 S rRNA. This structural feature was preserved and enhanced in 23 S rRNA after its association with the rest of the early assembly proteins, namely L3, L20, L13, L4 and L22. Since the above proteins, with the exception of L3, bind to the 5′ end of the 23 S rRNA, the cluster seems to be formed predominantly by interactions of L24, L13, L20, L22 and L4 with this segment of the 23 S rRNA molecule. Association with the rest of the primary binding proteins (L2, L23, L9, L1), which interact with the 3' end of the 23 S rRNA, appears to result in the formation of a second mass center. Binding of additional proteins led to the formation of compact particles with an apparent similarity to the 50 S subunit. However, particles with defined structural features characteristic of the native 50 S subunit requires the interactions of both 23 S rRNA and 5 S rRNA with all of the L-proteins. STEM image analysis demonstrated that 50 S subunit reconstitution proceeds by the immediate folding of the 23 S rRNA into a single mass center followed by the formation of a second mass center. These mass centers merge into one central body, which is gradually enhanced and decorated with structural elements characteristic of the 50 S subunit in the latter stages of assembly.

Scanning transmission electron microscopy (STEM) was used to visualize formation of a ternary complex between the T. thermophilus elongation factor (EF) Tu · GTP and the Escherichia coli Phe-tRNAPhe labeled with an undecagold (Au11) cluster at minor nucleotide 3-(3-amino-3-carboxypropyl) uridine at position 47. The ternary complex was further characterized by the molecular mass and radius of gyration calculated from the mass distribution within the individual particles. Under conditions used for STEM imaging, the ternary complex is formed between Au11-labeled Phe-tRNAPhe and Tu · GTP in a yield up to 25%. The stoichiometry of EF-Tu · GTP to aminoacyl-tRNA (aa-tRNA) in the EF-Tu · GTP · aa-tRNA complex is 1:1, in agreement with the established view of the protein biosynthesis mechanism. The ternary complex is also formed, although to a lower extent, with GTP analogues (GMPPCP and GMPPNP, respectively), but not with Tu · GDP and nonaminoacylated tRNAPhe with Tu · GTP.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the SOS monosomes and the large (608) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the “native” eukaryotic ribosome 3-D model.

The development of high resolution electron microscopic techniques and the introduction of quantitative image analysis has provided a direct basis for studying the structure of biological macromolecules. Apart from the determination of overall morphology, including 3-D image reconstruction, these techniques provide information on the internal architecture of biological macromolecular assemblies, the topography of functional sites, the interactions of their components and elemental microanalysis in situ. Thus, from a mere photographic recording system of the past, electron microscopy has evolved into a high resolution quantitative spectroscopic analytical method.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2G966 (m2G MT), while the other one makes m5C967 (m5C MT). The m2G MT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C MT functions reciprocally, using free rRNA but not 30S subunits (Negre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid Methylation (Alan Liss, New York), pp. 1-17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m5C MT, while simultaneously inducing methylation by the m2G MT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G MT but not by the m5C MT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G MT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-M., Craven, G. and Noller, H.F. (1988) J. Mol. Biol. 200, 309-319). Our results show that the two ribosomal RNA MTs studied in this work are exquisitely sensitive to this small but nevertheless functionally important structural change.

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 Å, and 990 kDa and 75 Å, respectively), which were greater than the theoretical values, while the activity was minimal (∼ 12%). The Mr and RG parameters of the 50S subunits remained uneffected by the NH4Cl washes (∼ 1600 kDa and 68 Å).

Separation of the resolution and contrast affecting components and the optimized placement of detectors for the collection of elastic (contrast-forming) electrons on Brookhaven dedicated STEM make it possible to quantitatively detect greater than 90% of the available elastically scattered electrons. The high contrast and superior signal-to-noise ratio associated with the STEM annular detector allow for the imaging of unstained freeze-dried biological macromolecular complexes (chromatin, viruses, nucleic acids) at radiation doses as low as 1 e/Å. Specimens prepared in this way are free of the main resolution-limiting conditions of conventional TEM i.e. staining , air drying and radiation damage. The image intensity of unstained specimens can be related to their local projected mass and used for calculation of the total mass and mass distribution within any selected particle. Elimination of staining makes it possible to use heavy metals as high-resolution markers for topographical mapping of components and/or functional sites on a particular macromolecular complex.

Scanning transmission electron microscopic images of transfer RNAs reveal the molecular dimensions and compact morphology of these small macromolecules in unprecedented detail. Selective labeling of a sulfhydryl group on 2-thiocytidine enzymatically inserted at position 75 at the 3′ end of yeast tRNAPhe, with an undecagold cluster permits identification of this specific tRNA site by dark field STEM. Imaging of a single nucleotide at a defined location on the tRNA molecule should make it possible to localize in situ tRNAs at the A, P, and E sites of the ribosomal peptidyl transferase center, and in complexes of tRNA with enzymes and elongation factors. In addition, this approach may be used for the highly specific topographical mapping of other RNAs and/or biological macromolecular complexes.

Protein-nucleic acid interactions involved in the assembly process of the Escherichia coli 30S ribosomal subunit were quantitatively analyzed by high-resolution scanning transmission electron microscopy. The in vitro reconstituted ribonucleoprotein (core) particles were characterized by their morphology, mass, and radii of gyration. During the assembly of the 30S subunit, the 16S rRNA underwent significant conformational changes that were governed by the cooperative interactions of the ribosomal proteins. The sequential association of the first 12 proteins with the 16S rRNA resulted in the formation of core particles containing up to three mass centers at distinct stages of the assembly process. These globular mass centers may correspond to the three major domains (5', central, and 3') of the 16S rRNA. Through the subsequent interactions of the late assembly proteins with the 16S rRNA, two of the three domains merge, yielding the basic structural traits of the native 30S subunit. The fine morphological features of the native 30S subunit became distinctly resolved only after the addition of the full complement of proteins. The fully reconstituted 30S subunits are active in polyphenylalanine synthesis assays. Visualization of the assembly mechanism of the E. coli 30S ribosomal subunit revealed domain-specific folding of the 16S rRNA through the formation of distinct intermediate core particles hitherto not observed.

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.

Presently, dedicated scanning transmission electron microscopy (STEM) is the most direct, most versatile and least destructive electron microscopic technique for high resolution studies of biological specimens. Apart from the morphology (shape, characteristic dimensions) of biological macromolecules, STEM can provide quantitative information on their molecular mass, the mass distribution within individual particles (for calculation of the radius of gyration), and the topography of their individual macromolecular components and functional sites. Deposition of nucleic acid molecules under non-denaturing conditions opened the EM field for studies of their conformation under various ionic conditions, their interactions with other macromolecules, proteins in particular, and ultimately to the visualization of the total reconstitution process of macromolecular complexes from their constituents.The potential of high resolution STEM at Brookhaven National Laboratory (BNL) was applied to the study of structure-function relationships of ribosomal particles of Escherichia coli, minute but complex cellular organelles playing a fundamental role in protein synthesis.

The secondary structure of HeLa 18S rRNA was investigated by a combination of chemical and enzymatic probing techniques. Using four chemical reagents (DMS*, kethoxal, DEPC and CMCT) which react specifically with unpaired bases and two nucleases (RNase T1 and cobra venom nuclease) which cleave the ribopolynucleotides at unpaired guanines and helical segments, we have analyzed the secondary structure of the 5' domain of 18S rRNA isolated from HeLa 40S ribosomal subunits. The sites at which chemical modifications and nuclease cleavages occurred were identified by primer extension using synthetic deoxyoligonucleotides and reverse transcriptase. These studies led to the deduction of an intra-RNA pairing pattern from the available secondary structure models based on comparative sequence analysis. Apart from the general canonical pairing we have identified noncanonical U-U, G-A, A-G, A-C, C-A and G-G pairing in HeLa 18S rRNA. The differential reactivity of bases to chemical reagents has enabled us to predict the possible configuration of these bases in some of the noncanonical pairing. The absence of chemical reactivities and cobra venom nuclease sensitivity in the terminal loops of helices 6 and 12 indicate a tertiary interaction unique to HeLa 18S rRNA. We have confirmed the existence of the complex tertiary folding recently proposed (Gutell and Woese 1990 Proc. Natl. Acad. Sci. 87, 663-667) for the universally conserved helix 19 in HeLa 18S rRna. The complementarity of chemical modifications and enzymatic cleavages provided experimental evidence for the proposal of a model structure for the 655 nucleotides of the 5' domain of HeLa 18S rRNA.

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.

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−. 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.

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.

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).