ABSTRACT
In the process of protein synthesis, the translating ribosomes of eukaryotic cells form polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic polyribosomes are circles (circular polyribosomes) and two-stranded formations (so-called double-row polyribosomes). The "long" (i.e. heavy loaded) polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular ("collapsed rings"), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in polyribosomes by marking them with gold nanoparticles (3'-end) and initiating 40S subunits (5'-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in polyribosomes and mutual orientation of ribosomes in them has shown that the double-row polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.
Subject(s)
Eukaryota/genetics , Polyribosomes/chemistry , RNA, Messenger/chemistry , Eukaryota/chemistry , Eukaryota/metabolism , Humans , Nucleic Acid Conformation , Polyribosomes/genetics , Polyribosomes/metabolism , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Messenger/metabolismABSTRACT
Binding of mRNA leader sequences to ribosomes was studied in conditions of a cell-free translation system based on wheat germ extract. Leader sequence of TMV mRNA (the so-called omega-RNA sequence) was able to bind simultaneously 80S ribosome and 40S ribosomal subunit. It was found that nucleotide substitutions in omega-RNA resulting in destabilization of RNA structure have no effect on the complex formation with both 80S ribosome and 40S ribosomal subunit. Leader sequence of globin mRNA is also able to form a similar joint complex. It is supposed that the ability of mRNA leader sequences to bind simultaneously 80S ribosome and 40S subunit is independent of leader nature and may reflect previously unknown eukaryotic mechanisms of translation initiation.
Subject(s)
5' Untranslated Regions , Eukaryota/genetics , Protein Biosynthesis , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Animals , Eukaryota/chemistry , Eukaryota/metabolism , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/geneticsABSTRACT
Formation of amyloid-like protein aggregates in human organs and tissues underlies many serious diseases, therefore being in the focus of numerous biochemical, medical, and molecular biological studies. So far, formation of amyloids by globular proteins has been studied mostly under conditions that strongly destabilized their native structure. Here we present our results obtained at permissive temperature by thioflavin T fluorescence, far UV CD, IR spectroscopy, and electron microscopy. We used apomyoglobin and its mutants with Ala or Phe substituted for Val10 that are structurally close to wild type apomyoglobin. It is shown that at permissive temperature the ability of the protein to form amyloids depends on the extent of its structural destabilization, but not on hydrophobicity of the substituting residue. A possible difference between amyloids formed by strongly destabilized proteins and those yielded by proteins with a slightly fluctuating native structure, as well as the stroke and infarction effect on the ability of proteins to form amyloid structures, are discussed.
Subject(s)
Amyloid/chemistry , Amyloid/genetics , Apoproteins/chemistry , Apoproteins/genetics , Myoglobin/chemistry , Myoglobin/genetics , Point Mutation , Valine/genetics , Amyloid/metabolism , Apoproteins/metabolism , Circular Dichroism , Humans , Myoglobin/metabolism , Protein Conformation , Protein Folding , Protein Stability , Temperature , Valine/chemistry , Valine/metabolismABSTRACT
Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Endotoxins/chemistry , Insecticides/chemistry , Liposomes/chemistry , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , Coleoptera/drug effects , Endotoxins/pharmacology , Hemolysin Proteins , Hot Temperature , Insecticides/pharmacology , Lipid Bilayers , Microscopy, Electron , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Phospholipids/chemistry , Protein Denaturation , ThermodynamicsABSTRACT
A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five T. thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.
Subject(s)
RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/genetics , Base Sequence , Escherichia coli , Microscopy, Electron , Molecular Sequence Data , Nucleic Acid Conformation , Protein Folding , RNA, Bacterial/ultrastructure , RNA, Ribosomal/chemistry , RNA, Ribosomal/ultrastructure , RNA, Ribosomal, 16S/ultrastructure , Ribosomal Proteins/ultrastructure , Thermus thermophilus/ultrastructure , Transcription, GeneticABSTRACT
Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies.
Subject(s)
Ribosomes/chemistry , Thermus thermophilus/chemistry , Bacterial Proteins/chemistry , Base Sequence , Crystallography , Microscopy, Electron , Molecular Sequence Data , Neutrons , Nucleic Acid Conformation , Protein Biosynthesis , RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Ribosomal Proteins/chemistry , Ribosomes/ultrastructure , Thermus thermophilus/genetics , Thermus thermophilus/ultrastructure , X-Ray DiffractionABSTRACT
A working model of the mRNA path through the ribosome is proposed. According to the model, the template goes around the small ribosomal subunit along the region where its 'head' is separated from other parts of the subunit. The 5'-end of the mRNA fragment covered by the ribosome is located near the 3'-terminus of 16S rRNA, whereas the 3'-terminal residues of the fragment are situated on the outer surface of the subunit, opposite its 'side ledge'. When associated with the 50S subunit, the 30S subunit is oriented in such a manner that the decoding center faces the L7/L12 stalk. Implications of the proposed working model of the mRNA topography for the function of the ribosome are discussed.
Subject(s)
RNA, Messenger/metabolism , Ribosomes/metabolism , Binding Sites , Microscopy, Electron , Models, Biological , RNA, Messenger/chemistry , RNA, Messenger/ultrastructure , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/metabolism , Ribosomes/ultrastructureABSTRACT
Human immunoglobulin G1 Van was studied by negative staining, freeze drying and high resolution shadow casting. The Fab and Fc subunits of an intact IgG1 molecule were shown to possess limited mobility. It was found that about 70% of molecules in the IgG1 Van specimen are not flat but have a tripod-like shape.
Subject(s)
Immunoglobulin G/ultrastructure , Humans , Immunoglobulin Fab Fragments/ultrastructure , Microscopy, Electron , Myeloma Proteins , Protein ConformationABSTRACT
Small ribosomal subunits from rat liver have been studied by electron microscopy using freeze-drying and high-resolution shadow casting. The absolute hand of the asymmetric subunit has been determined and its three-dimensional model with a 'right' location of the side protuberance has been constructed. The results evidence that pro- and eukaryotic ribosomes have a unique and principally similar structural organization.
Subject(s)
Liver/ultrastructure , Ribosomal Proteins/ultrastructure , Animals , Freeze Drying , Microscopy, Electron/methods , Molecular Structure , RatsABSTRACT
Non-precipitating anti-dinitrophenyl pig immunoglobulins G have been studied by negative staining, freeze-drying and high-resolution shadow casting. The general morphology of the molecules is described. The predominant conformation of antibody molecules is a tripod-like one.
Subject(s)
Dinitrophenols/immunology , Immunoglobulin G , Animals , Freeze Drying , Immunoglobulin G/isolation & purification , Microscopy, Electron , Protein Conformation , SwineABSTRACT
Immuno-electron microscopy studies on the morphology of the Escherichia coli ribosome and the topography of its functional centers are summarized. A three-dimensional model of the ribosome with indications of the mRNA-binding site, the peptidyl-transferase center, the EF-Tu and EF-G-binding sites and the tRNA-binding sites in given. Thus, the mutual arrangement of the centers corresponding to the main functional activities of the ribosome during the process of translation is presented.
Subject(s)
Cells/ultrastructure , Prokaryotic Cells/ultrastructure , Ribosomes/metabolism , Binding Sites , Microscopy, Electron , Peptide Elongation Factors/metabolism , Peptidyl Transferases/metabolism , Prokaryotic Cells/metabolism , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosomes/ultrastructureABSTRACT
Purified preparations of Q beta replicase have been studied by electron microscopy using a negative staining technique, and a three-dimensional model of the enzyme molecule has been constructed. The molecule of this four-subunit protein appears to be a compact structure having a size of 100 +/- 10 A; it is subdivided into two unequal bipartite subparticles. The conclusion has been made that all the constituent subunits, including the ribosomal protein Sl, acquire a globular conformation when associated in the replicase complex.
Subject(s)
Coliphages/enzymology , Escherichia coli/enzymology , Q beta Replicase , RNA Nucleotidyltransferases , Microscopy, Electron , Protein ConformationSubject(s)
RNA, Ribosomal/ultrastructure , Centrifugation, Density Gradient/methods , Indicators and Reagents , Microscopy, Electron/methods , RNA, Ribosomal, 16S/isolation & purification , RNA, Ribosomal, 16S/ultrastructure , RNA, Ribosomal, 23S/isolation & purification , RNA, Ribosomal, 23S/ultrastructure , Ribosomes/ultrastructureABSTRACT
MS2 phage RNA-directed synthesis of an N-terminal polypeptide of the phage coat protein on Escherichia coli 70 S ribosomes was initiated in a cell-free system with the N-dinitrophenyl derivative of methionyl-tRNAFMet) and performed in the absence of tyrosine, lysine, cysteine and methionine. As a result, the translating ribosomes carried peptides up to 42 amino acid residues in length with the dinitrophenyl hapten at the N-ends. Using the immune electron microscopy technique the positions of the nascent peptide N-ends on the 70 S ribosomes have been visualized. It has been found that (i) the N-ends of nascent peptides of these lengths are accessible to antibodies, (ii) the exit site of a nascent peptide is the pocket between the base of the central protuberance and the L1 ridge on the 50 S subunit, i.e. presumably its peptidyl transferase center, and (iii) the further pathway of a nascent peptide seems to proceed along the groove on the external surface of the 50 S subunit.
Subject(s)
Escherichia coli/metabolism , Protein Biosynthesis , Ribosomes/metabolism , Antibodies , Antigen-Antibody Complex/analysis , Cell-Free System , Coliphages/genetics , Coliphages/metabolism , Escherichia coli/genetics , Escherichia coli/ultrastructure , Microscopy, Electron , Peptide Biosynthesis , Ribosomes/ultrastructureABSTRACT
The EF-Tu-binding center of the E. coli ribosome has been localized by immunoelectron microscopy after cross-linking of the specific EF-Tu X 70 S ribosomal complex with dimethylsuberimidate. EF-Tu has been found to be in contact with the 50 S subunit in the region of the L7/L12 stalk and with the 30 S subunit in the upper part of its body on the side opposite the top of the ledge (the platform). The EF-Tu position on a model of the 70 S ribosome is presented.
Subject(s)
Escherichia coli/ultrastructure , Peptide Elongation Factor Tu/metabolism , Ribosomes/metabolism , Binding Sites, Antibody , Cross-Linking Reagents/pharmacology , Dimethyl Suberimidate/pharmacology , Immunologic Techniques , Macromolecular Substances , Microscopy, Electron , Models, Structural , Peptide Elongation Factor Tu/immunology , Ribosomes/immunologyABSTRACT
The complex of 23 S ribosomal RNA with the nine core proteins L2, L3, L4, L13, L17, L20, L21, L22 and L23 obtained either by the disassembly procedure or by reconstitution has been studied by electron microscopy. This complex is found to be very similar to the intact 50 S subunit both in size and in shape.
Subject(s)
Escherichia coli/analysis , RNA, Bacterial , RNA, Ribosomal , Ribonucleoproteins , Centrifugation, Density Gradient , Chemical Phenomena , Chemistry, Physical , Chlorides/pharmacology , Ethanol/pharmacology , Lithium/pharmacology , Lithium Chloride , Microscopy, Electron , Nucleic Acid Conformation/drug effects , Spermidine/pharmacologyABSTRACT
50 S ribosomal subunit derivatives without the 5 S RNA-protein complex obtained either by splitting with EDTA or by reconstitution from the 23 S RNA and proteins have been studied by electron microscopy. Removal of the 5 S RNA-protein complex is shown to affect neither the overall morphology of the larger ribosomal subunit nor the mode of its association with the small subunit.
Subject(s)
Escherichia coli/analysis , RNA, Bacterial , RNA, Ribosomal , Ribonucleoproteins , Centrifugation, Density Gradient , Edetic Acid/pharmacology , Macromolecular Substances , Microscopy, ElectronABSTRACT
Elongated hollow strands were revealed on raw images and averaged by the correlation method images of the 30 S subunit of the E. coli ribosome negatively stained by uranyl acetate. The tentative three-dimensional arrangement of the 'strands' and their nature are discussed.
Subject(s)
Escherichia coli/ultrastructure , Ribosomes/ultrastructure , Microscopy, Electron , Models, StructuralABSTRACT
The internal site of 5 S RNA comprising residues A39 and U40 has been localized on the E. coli 50 S ribosomal subunit by immune electron microscopy. It has been found to be located on the interface side of the central protuberance at the position distinctly apart but very close to the position of the 5 S RNA 3'-end providing evidence for a quite compact folded conformation of the 5 S RNA in situ.
Subject(s)
Escherichia coli/analysis , RNA, Bacterial/analysis , Ribosomes/analysis , Centrifugation, Density Gradient , Chemical Phenomena , Chemistry , Electrophoresis, Polyacrylamide Gel , Endonucleases/metabolism , Ethylenediamines , Haptens , Immunologic Techniques , Macromolecular Substances , Microscopy, Electron , RNA, Bacterial/metabolism , Single-Strand Specific DNA and RNA EndonucleasesABSTRACT
Freeze-dried and shadowed Escherichia coli 50 S ribosomal subunits have been examined by electron microscopy and a model of the subunit has been constructed. High resolution shadow casting has enabled us to determine independently the absolute hand of the subunit and to reveal some new structural features.