Mitochondrial and cytoplasmic ribosomes and their activity in blood and culture form Trypanosoma brucei

Ribosomes of Trypanosoma brucei, a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers. Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes. These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg-++ concentrations, dissociating below 0.1 mM Mg++. The 72S ribosomes were more sensitive to elevated KCl concentrations, dissociation above 0.25 M. Protein synthetic activity associated with the 72S class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.     

Protein synthesizing cell apparatus in malignant growth and pharmacotherapy by chlofiden

Data on the influence of a new antitumor preparation chlofiden on the general contents of rat liver ribosomes and sarcoma 45 and their division on free and membrane of membrane bound and decrease of free ribosomes during tumor growth supposed synthesis of specific proteins bound are given in the paper. It was shown that in the liver of tumor bearing rats total and membrane bound ribosomes decreased and the level of free ribosomes increased. High contents of free ribosomes in sarcoma 45 may testify increase of intracellular protein synthesis including processes of cell growth and division as well as the tendency for increase. Chlofiden normalized total contents, increased free and decreased liver membrane bound ribosomes contents, during tumor growth supposed synthesis of specific proteins. Increase of free ribosomes and decrease of their specific radioactivity in sarcoma 45 testified membrane damage by chlofiden and inhibition of intracellular protein synthesis which are essential in cell division.     

Dormant ribosomes inBlastocladiella emersonii zoospores are arrested at elongation

Ribosomes fromBlastocladiella emersonii zoospores stimulatein vitro protein synthesis in a system using soluble factors extracted from wheat germ. Aurintricar☐ylic acid inhibits less than 40% of thein vitro protein synthetic activity of the zoospore ribosomes, indicating that messenger RNA is already complexed to the ribosomes. In addition to the mRNA complexed to the ribosomes, zoospores contain an mRNA fraction which is not bound to the ribosomes. Extraction of RNA from zoospore ribosomes and deacylation followed by reacylation with labeled amino acids demonstrated the presence of tRNA molecules specific for methionine and other amino acids on zoospore ribosomes. Transfer RNA from zoospore ribosomes contained 9.8% methionyl-tRNA compared to 2.4% methionyl-tRNA bound to ribosomes isolated from growth-phase plants. The fourfold enrichment of methionyl-tRNA on zoospore ribosomes suggests that between 12 and 25% of the zoospore ribosomes exist in arrested 80 S initiation complexes. Collectively, the data indicate that zoospore ribosomes complexed to mRNA have completed initiation and are somehow blocked at one or more of the elongation steps of protein synthesis. The data are compatible with the idea that an inhibitor is associated with the zoospore ribosomesin vivo.     

Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth

Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with ¹⁴C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20–25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10–15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.     

Properties of human mitochondrial ribosomes

Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.     

Experimental changes in the amount of maternally stored ribosomes affect the translation efficiency of ribosomal protein mRNA in Xenopus embryo

The amount of maternal free ribosomes in developing Xenopus embryos has been experimentally modified; an increase was obtained by microinjection of purified ribosomes into fertilized eggs, and a decrease was induced by treatment with a drug which reduces the amount of free ribosomes. The effect of this manipulation on the partition of the ribosomal protein mRNA (rp-mRNA) was analyzed during embryo development; it was observed that when ribosomes available for translation are in excess, polysome loading with rp-mRNA decreases. Conversely, when ribosomes are scarce, polysome loading of rp-mRNA increases. These experiments, which artificially stress events observed in the course of development, indicate that there is a relationship between the availability of ribosomes in the cells and the utilization of rp-mRNA for synthesis of ribosomal proteins, as already suggested by previous observations on r-protein synthesis during embryogenesis.     

Ribosomes from a relC mutant strain of Escherichia coli show altered activity with bacterial release factor-1

Ribosomes from a relC mutant of Escherichia coli, JF505, are altered in the large subunit protein L11. This protein has abnormal mobility on gel electrophoresis. The ribosomes have a lowered specific activity for release factor-1 which is intermediate between that found for ribosomes containing normal L11 and that for L11 lacking ribosomes. JF505 ribosomes are as sensitive to inactivation of in vitro termination by thiostrepton as normal ribosomes when the antibiotic is added in dimethylsulphoxide but less sensitive when it is added in ethanol.     

Proteins of mammalian mitochondrial ribosomes.

The bovine mitochondrial system is being developed as a model system for studies on mammalian mitochondrial ribosomes. Information is emerging on the structural organization and RNA binding properties of proteins in these mitochondrial ribosomes. Unexpectedly, these ribosomes appear to interact directly with GTP, via a high affinity binding site on the small subunit. Despite major differences in their RNA content and physical properties, mammalian mitochondrial and cytoplasmic ribosomes contain about the same number of proteins. The proteins in each kind of ribosome have a similar size distribution, and both sets are entirely coded by nuclear genes, raising the possibility that these different ribosomes may contain the same set of proteins. Comparison of bovine mitochondrial and cytoplasmic r-proteins by co-electrophoresis in two-dimensional gels reveals that most of the cytoplasmic ribosomal proteins are more basic than the mitochondrial ribosomal proteins, and that none are co-migratory with mitochondrial ribosomal proteins, suggesting that the proteins in the two ribosomes are different. To exclude the possibility that the electrophoretic differences result only from post-translational modification of otherwise identical proteins, antibodies against several proteins from the large subunit of bovine mitochondrial ribosomes were tested against cytoplasmic ribosomes by solid phase radioimmunoassay and against cytoplasmic ribosomal proteins on Western blots. The lack of cross-reaction of these antibodies with cytoplasmic r-proteins suggests that mitochondrial ribosomal proteins have different primary structures and thus are most likely encoded by a separate set of nuclear genes.     

Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins

Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates.     

Effect of ribosome treatment on sensitivity to ricin A chain

Rat liver ribosomes treated with catalytic amounts (30 ng/ml) of ricin A chain are inhibited about 80% when assayed immediately. However, the same ribosomes assayed after separation from A chain by centrifugation have partially recovered their activity in the translation of polyuridylic acid. The extent of recovery is dependent on magnesium ion concentration. Even though the activity of A chain-treated ribosomes is increased after centrifugation, they are not sensitive to further treatment with ricin A chain. Except for impure ribosomes, isolated by centrifugation of crude homogenate, the overall sensitivity of ribosomes after different treatments was the same.     

Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis

On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed. There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid. Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U] in the incubation mixture. Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA. The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs. These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis. The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process. A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples.     

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.     

Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3)

Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content. However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes. To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes. Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes. Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction. Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.     

Cell-free protein synthesis in t,e rabbit liver ribosomal system. II. Large scale preparation of purified 80S ribosomes.

A procedure for the preparation of a large quantity of biologically active, highly purified ribosomes from rabbit liver is described. The method employs polyethylene glycol-dextran sulfate parition and DEAE-cellulose chromatography to overcome the limitations encountered in conventional procedures. The entire process takes only 48 h to obtain 10,000 A(260) units of ribosomes. The ribosomes thus obtained are predominantly 78S particles with a constant protein-RNA ratio of 0.95. The ribosomes are free from RNase, amino-acyl-tRNA synthetase, and amino-acyl-tRNA: protein transferase activity. The protein synthesizing activity is dependent on added mRNA and protein factors. These ribosomes are stable for prolonged periods of storage in a liquid nitrogen refrigerator.     

Immunological comparison of the proteins of chicken and rat liver ribosomes.

A comparison of the proteins of chicken and rat liver ribosomes using immunochemical techniques was undertaken. The procedures included quantitative precipitation, passive hemagglutination, and immunodiffusion on Ouchterlony plates. The results indicate that antisera specific for chicken or rat liver ribosomes recognize only about 20% of common determinants. While there are important reservations, the results suggest extensive differences in the proteins of rat and chicken liver ribosomes. Despite those differences, rat and chicken liver ribosomal proteins maintain some homologous sequences present in bacterial ribosomal proteins. An enriched antibody preparation against chicken 80 S ribosomes inhibited the poly(U)-directed synthesis of polyphenylalanine and the elongation factor G (EF-G)-catalyzed binding of [3H]GDP to Escherichia coli ribosomes. Thus, chicken liver ribosomes, like ribosomes from rat liver and yeast, must have proteins homologous with those of E. coli ribosomes.     

Role of ribosomes in cycloheximide resistance of Neurospora mutants

Summary In Neurospora crassa, mutants resistant against cycloheximide appear with a marked time lag after mutation induction. We have suggested (Neuhäuser et al., 1970) that this lag indicates the time needed for the synthesis of altered ribosomes (phenotypic lag), that the drug in the wildtype acts upon the ribosomes, and that resistance is due to alterations in them.By measurements of poly-U directed poly-Phe synthesis on ribosomes of the wildtype and two different cycloheximide resistant mutants in a cell free system it is shown here that mutant ribosomes indeed differ from those of the wildtype. Poly-Phe synthesis on mutant ribosomes proceeds in the presence of the drug, whereas that on wildtype ribosomes is inhibited. This means that the earlier suggestions are correct.Abbreviation CHX cycloheximide     

ELECTRON MICROSCOPE STUDY OF MITOCHONDRIAL 60S AND CYTOPLASMIC 80S RIBOSOMES FROM LOCUSTA MIGRATORIA

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl₂. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of ~270 x 210 x 215 Å; cytoplasmic ribosomes measure ~295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is ~130 Å, while the diameter of cytoplasmic ribosomes is ~ 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.     

Evolution of a protein-rich mitochondrial ribosome: implications for human genetic disease

Mitochondrial ribosomes comprise the most diverse group of ribosomes known. The mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. The bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Mammalian mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system. Interest is growing in the structure, organization, chromosomal location and expression of genes for human MRPs. Proteins which are essential for mitoribosome function are candidates for involvement in human genetic disease.     

Structural-functional topography of human ribosomes based on the data from crosslinking with mRNA analogs--oligoribonucleotide derivatives

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable group at different positions. A comparison of data on template position on the human and Escherichia coli ribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A, P, and E sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem-loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.