Localization of the site of cleavage of ribosomal RNA by colicin E3. Placement on the small ribosomal subunit by electron microscopy of antibody--complementary oligodeoxynucleotide complexes

Colicin E3 is a ribonuclease that inactivates Escherichia coli ribosomes by cleaving the RNA of the small ribosomal subunit after nucleotide 1493. A series of oligodeoxynucleotides that complement 16 S RNA in the region of the colicin cleavage site has been synthesized, and their ability to form complexes with 30 S ribosomal subunits has been measured using a nitrocellulose filter-binding assay. The most efficiently bound probe, complementary to residues 1485-1496, was modified with antibody-recognizable derivatives at the 5'-end, the 3'-end, or both. Antibody-oligonucleotide-subunit complexes were then generated and examined by electron microscopy. Antibody binding was seen at the tip of the platform of the 30 S subunit. The complementary oligonucleotide and thus the site at which colcin E3 cleavage occurs is therefore in the same physical region as the 3'-end of the 16 S ribosomal RNA and its message-positioning "Shine-Dal-garno" sequence.     

Messenger RNA movement on the ribosome

The review summarizes the recent structural data obtained for 70S ribosome complexes with various mRNAs and tRNAs by X-ray analysis and cryoelectron microscopy. The mRNA region interacting with the ribosome at translation initiation and elongation is described. A specific part (platform) of the 30S ribosome subunit was assumed to bind the regulatory elements located in the 5′-untranslated region of mRNA.     

Messenger RNA movement on the ribosome

Recent X-ray and cryo-EM studies of 70S ribosome complexes containing different types of messenger RNAs (mRNA) and transfer RNA (tRNA) have been reviewed. Changes of the mRNA path on the ribosome at initiation and elongation states have been described. Authors suggested, that the specific region of ribosomal 30S subunit ("platform") is a ribosome binding site of regulatory domains of mRNA which locates on the non-translated 5'-end of the mRNA.     

Electron microscopy of functional ribosome complexes

Cryoelectron microscopy has made a number of significant contributions to our understanding of the translation process. The method of single-particle reconstruction is particularly well suited for the study of the dynamics of ribosome-ligand interactions. This review follows the events of the functional cycle and discusses the findings in the context provided by the recently published x-ray structures.     

Relative orientation of RNA helices in a group 1 ribozyme determined by helix extension electron microscopy.

The relative orientation of helical elements in a folded RNA molecule provides key information about its three-dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/- 20 degrees angle, consistent with the X-ray structure. Single, double and triple extensions (50-80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel-Westhof structural model, while the P2.1-P6b and P2.1-P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low-resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.     

Localization of an oligodeoxynucleotide complementing 16S ribosomal RNA residues 520-531 on the small subunit of Escherichia coli ribosomes: electron microscopy of ribosome-cDNA-antibody complexes.

The oligodeoxynucleotide dACCGCGGCTGCT, complementary to Escherichia coli small ribosomal subunit RNA residues 520-531, has been used to probe subunit conformation and to localize the sequence in the subunit. Conditions for binding of the cDNA to 30S subunits were optimized and specificity of the interaction was demonstrated by RNase H cleavage. Three kinds of terminal modification of this cDNA were used to allow its localization by immune electron microscopy. A solid phase support with 5'-dimethoxytrity-N6-delta 2-isopentenyl-adenosine linked to controlled pore glass was synthesized, and used to prepare oligomer with an added 3'-terminal residue of isopentenyl adenosine. cDNA with a 5' primary amine substituent was modified with 1-fluoro-2,4-dinitrobenzene to prepare 5'-dinitrophenyl oligonucleotide, and both modifications together gave doubly-derivatized probes. Immune electron microscopy with antibodies to dinitrophenol, isopentenyl adenosine, or both, was used to place the cDNA on 30S subunits. In each case the probe was placed at a single site at the junction of the head and body of the subunit, near the decoding site and the area in which elongation factor Tu is bound. It is proposed that this segment of ribosomal RNA functions in mRNA binding and orientation.     

Demonstration of immune complexes on erythrocytes by scanning electron microscopy

If anti-sera are combined with native whole blood by an in-vitro technique, immunocomplexes are formed. They are fixed to erythrocytes and can be made visible by a scanning electron microscope on blood smears especially pretreated.     

Nucleoprotein-RNA orientation in the measles virus nucleocapsid by three-dimensional electron microscopy

Recombinant measles virus nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer.     

Visualization of DNA and RNA molecules, and protein-DNA complexes for electron microscopy

This article provides step-by step instructions for the preparation of double- and single-stranded DNA and RNA molecules and protein-DNA complexes for electron microscopy (EM). Absorption, spreading, staining, dark-field imaging, and metal shadowing techniques are described in detail. A number of examples are illustrated on analysis of DNA replication, DNA repair and DNA recombination to demonstrate the usefulness of the technique for EM visualisation. Application of immunogold labeling of specific protein in DNA-protein complexes is also covered.     

Specific visualization of ribosomal RNA in the intact ribosome by electron spectroscopic imaging

The recently developed electron microscopic technique of electron spectroscopic imaging has been used to map the distribution of phosphorus, and therefore of RNA, in situ in the ribosomal subunits of E. coli. The results indicate that the RNA moiety of both subunits is concentrated toward the centre of the particle somewhat more than is the total mass, but reaches the outer surface at several places. The micrographs also reveal certain distinctive features in the shape of the RNA component that may be related to the overall shape of the ribosome. The method yielded a reasonably accurate estimate of the phosphorus content of the 30 S ribosome.     

Visualizing nuclear export of different classes of RNA by electron microscopy.

Export of RNA from the cell nucleus to the cytoplasm occurs through nuclear pore complexes (NPCs). To examine nuclear export of RNA, we have gold-labeled different types of RNA (i.e., mRNA, tRNA, U snRNAs), and followed their export by electron microscopy (EM) after their microinjection into Xenopus oocyte nuclei. By changing the polarity of the negatively charged colloidal gold, complexes with mRNA, tRNA, and U1 snRNA can be formed efficiently, and gold-tagged RNAs are exported to the cytoplasm with kinetics and specific saturation behavior similar to that of unlabeled RNAs. U6 snRNA conjugates, in contrast, remain in the nucleus, as does naked U6 snRNA. During export, RNA-gold was found distributed along the central axis of the NPC, within the nuclear basket, or accumulated at the nuclear and cytoplasmic periphery of the central gated channel, but not associated with the cytoplasmic fibrils. In an attempt to identify the initial NPC docking site(s) for RNA, we have explored various conditions that either yield docking of import ligands to the NPC or inhibit the export of nuclear RNAs. Surprisingly, we failed to observe docking of RNA destined for export at the nuclear periphery of the NPC under any of these conditions. Instead, each condition in which export of any of the RNA-gold conjugates was inhibited caused accumulation of gold particles scattered uniformly throughout the nucleoplasm. These results point to the existence of steps in export involving mobilization of the export substrate from the nucleoplasm to the NPC.     

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.     

Messenger RNA conformation and ribosome selection of translational reinitiation sites in the lac repressor mRNA

In the early region of the Escherichia coli lac repressor mRNA, the pattern of cleavage by nucleases specific for single or double-stranded RNA confirms the presence of secondary structures previously proposed to influence the pattern of translational reinitiation. These are positioned so as to mask a potential restart site centered on an in-phase GUG triplet corresponding to repressor amino acid position Val38. Our finding that a restart polypeptide initiated at the Val38 GUG codon is observed only in situations that that preclude base-pairing of adjacent mRNA sequences establishes a functional role for these structures in vivo. This evidence for structure, considered with the overall pattern of reinitiation events, suggests that local mRNA conformation is the major determinant that dictates ribosomal selection of restart sites within the early region of the repressor cistron.     

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy.

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10-6 plus or minus 0.25 times 10-6.