Protein synthesis by single ribosomes

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Protein-Synthetic Activity of Ribosomes from Interferon-Treated Cells

Cell-free protein-synthetic systems from normal and interferon-treated chick cells were compared. No difference was found in the amino acid incorporation activities of such ribosome-cell sap systems or in their response to polyuridylic acid. Throughout a variety of experiments we failed to detect the formation of a discrete peak of virus-specific polysomes, when ribosome monomers and subunits (from interferon-treated or control cells) were incubated with labeled Sindbis or Semliki Forest virus ribonucleic acid (RNA). Some binding of viral RNA did occur, but the complexes formed were evident in sucrose gradients as a broad, rapidly sedimenting shoulder on the ribosome monomer peak. Interferon pretreatment of cells did not affect the formation of these complexes in vitro, nor did it alter their rate of breakdown on incubation under amino acid incorporation conditions. Experiments with inhibitors of protein synthesis showed that such "breakdown" was not dependent upon amino acid incorporation and was not an index of translation. In these respects, our results are in marked contrast to those of Marcus and Salb. These results, together with our failure to detect any significant change in the protein composition of ribosomes from interferon-treated cells, suggest that such treatment does not result in a modification of the ribosome per se. They do not, however, rule out the involvement of a factor(s) required for ribosomes and viral RNA to function in viral protein synthesis. Indeed, it remains likely that interferon acts through such a mechanism, although the precise level at which the inhibition occurs remains to be elucidated.     

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.     

Regulation of Protein Synthesis in Zoospores of Blastocladiella

The factors responsible for the regulation of protein synthesis in the zoospores of Blastocladiella emersonii were studied by means of cell fractionation and in vitro assays. Charged transfer ribonucleic acid (tRNA) and aminoacyl-tRNA synthetases were found both inside the membrane-bound, ribosomal nuclear cap, and in the extracap cytoplasm. Ribosomes isolated from zoospore nuclear caps in low salt buffer failed to support polyuridylic acid-dependent phenylalanine incorporation. After washing with high salt buffer, the cap ribosomes were equivalent in activity to similarly prepared plant ribosomes. Both the high-salt wash from cap ribosomes and the extracap supernatant fraction contained an unidentified material which inhibited aminoacyl-tRNA binding and peptide bond formation by ribosomes. Ribosomal binding of polyuridylic acid was not inhibited. Washed cap ribosomes supported very low incorporation rates without added messenger RNA, and were highly dependent upon added poly U for phenylalanine incorporation, indicating a low level of messenger in nuclear caps. It is concluded that enclosure of the ribosomes in the nuclear cap does not in itself prevent protein synthesis, and that the lack of activity may be due to the presence of a ribosome inhibitor.     

Protein Synthesis in a Cell-Free Extract from Staphylococcus aureus

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Translational control of protein synthesis in stimulated WI-38 fibroblasts.

A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgC12 and KC1 levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([14C]leucine incorporation/10 min/100 mug rRNA) and the number of growing peptide chains [3H]puromycinpeptides formed/100 mug rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 mug/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function.     

In vitro Protein Synthesis by Polyribosomes of Peach Flower Buds at Different Physiological Stages

The in vitro phenylalanine incorporation by polyribosomes of peach flower buds (Prunus persica Stokes) during dormancy, dormancy break and flowering was investigated. Protein synthesis was measured using as catalyst either calf liver soluble factors or the ribosomal supernatant from the peach flower buds in the presence or the absence of the synthetic mRNA, polyuridylic acid. In the presence of polyuridylic acid, the activity of protein synthesis of dormant ribosomes is the same as that of ribosomes during dormancy break and flowering. The absence of synthetic messenger did not cause a change in activity. The ribosomal supernatant of dormant buds, but not of flowering buds, reduces the phenylalanine incorporation by polyribosomes from buds harvested at dormancy break.     

Effect of food deprivation and hypophysectomy on in vitro protein synthesis by membrain-bound and free hepatic ribosomes.

The protein synthetic activities of membrane-bound and free hepatic ribosomes isolated from intact rats fed ad libitum, and normal rats subjected to food restriction to match that of hypophysectomised (Hx) rats were compared to the in vitro protein synthetic capacity of hepatic ribosomes isolated from Hx rats. Hypophysectomy resulted in decreased protein synthetic ability of bound ribosomes, whether protein synthesis was directed by endogenous messenger RNA (mRNA) (p less than 0.05) or by polyuridylic acid (polyU) (p less than 0.01). In contrast, the protein synthetic activity of free hepatic ribosomes from Hx rats was reduced when protein synthesis was directed by endogenous mRNA (p less than 0.05) but, when polyU was substituted as the messenger, the protein synthetic activity of these free ribosomes was equal to that of control rats. On the other hand the effects of food restriction on hepatic ribosomal function could be clearly differentiated from the effects observed following hypophysectomy. Thus, the reduced protein synthetic activity of hepatic bound ribosomes isolated from food restricted normal rats was not demonstrable, when polyU was used to direct protein synthesis. Further, food restriction had no effect on the protein synthetic activity of free hepatic ribosomes, and this was true when protein synthesis was directed by either endogenous or artificial messenger. It is concluded that hypophysectomy reduces the protein synthetic ability of both bound and free hepatic ribosomes, and this change of ribosomal function of Hx rats cannot be attributed to their decreased food intake.     

Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.     

Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system. J. Bacteriol. 92:197-203. 1966.-When tetracycline, an inhibitor of cell-free protein synthesis, was preincubated with each component of the Escherichia coli cell-free system, i.e., ribosomes, soluble ribonucleic acid (sRNA), polyuridylic acid (poly U), and S-100 (supernatant enzymes), only the ribosomal-bound antibiotic was inhibitory to the cell-free assay. Experiments designed to further localize the site of inhibition to either the 50S (Svedberg) or the 30S ribosomal subunit were not conclusive. Tritiated tetracycline (7-H(3)-tetracycline) was bound to isolated 50S ribosomes, and these were recombined with 30S subunits to form 70S ribosomes. When these ribosomes were dissociated and the subunits reisolated, the antibiotic was found with both the 50S and the 30S particles. The same results were observed when the tetracycline was initially bound to the 30S subunit.     

Protein synthesis by a cell-free preparation from the bird malaria, Plasmodium lophurae.

Cytoplasmic polyribosomes were isolated from the avian malaria parasite Plasmodium lophurae by lysis with 0.15% Triton X-100 followed by high speed centrifugation through a discontinuous sucrose gradient. Polyribosomes were protected from nuclease degradation using 100 mug/ml heparin or 50 mug/ml dextran sulfate. Cell-free incorporation of radioisotope-labeled amino acids required a pH 5 fraction (duck reticulocyte), Mg2+, and an energy-generating system. The protein synthesizing system was stimulated by the addition of polyuridylic acid. Optimum conditions for protein synthesis by the plasmodial system are described. The effects of drugs on the cell-free protein synthesizing system using duck reticulocyte and plasmodial ribosomes are reported.     

Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.

An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.     

The effect of morphine tolerance and dependence on cell free protein synthesis

A cell free system consisting of polyribosomes and pH 5 factors of the cytosol was isolated from mouse brain. This system actively promoted the incorporation of radiolabeled amino acids into protein in vitro. Addition of exogenous morphine to a cell free protein synthetic system isolated from chronically morphinized, placebo treated, or naive mouse brain had no effect on the relative synthetic capacity of the system. In addition, morphine did not alter the response to a synthetic mRNA, polyuridylic acid. However, both the polyribosomes and pH 5 factors isolated from chronically morphinized mouse brain were more effective in promoting amino acid incorporation into protein relative to the corresponding fractions from placebo treated mice. Acrylamide gel electrophoresis of the proteins in the incubation mixture showed the increased amino acid incorporation was the result of a general quantitative increase in the specific activity of all of the proteins synthesized by the cell free system.     

The roles of RNA in the synthesis of protein

The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding.     

Reduced turnover of the elongation factor EF-1 X ribosome complex after treatment with the protein synthesis inhibitor II from barley seeds

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [14C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 X GTP X aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.     

Inhibition of protein synthesis by polypeptide antibiotics. I. Inhibition in intact bacteria

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. I. Inhibition in intact bacteria. J. Bacteriol. 90:1102-1108. 1965.-The mechanism of inhibition of growth of cells by the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes was studied. This inhibition apparently was due to the selective inhibition of protein synthesis by these antibiotics. Ribonucleic acid synthesis was unaffected by concentrations of the antibiotics which completely inhibited protein synthesis. Deoxyribonucleic acid synthesis was slightly inhibited. These antibiotics are composed of a number of components. Mixtures of equal amounts of PA 114 A and PA 114 B or vernamycin A and Balpha were more active in stopping protein synthesis in intact cells than each of the components of the antibiotic complex alone. Mutants resistant to one of the antibiotics were resistant to all of the group and, in addition, were resistant to erythromycin and oleandomycin.     

Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

Elongation factors in protein biosynthesis

Translation elongation factors are the workhorses of protein synthesis on the ribosome. They assist in elongating the nascent polypeptide chain by one amino acid at a time. The general biochemical outline of the translation elongation cycle is well preserved in all biological kingdoms. Recently, there has been structural insight into the effects of antibiotics on elongation. These structures provide a scaffold for understanding the biological function of elongation factors before high-resolution structures of such factors in complex with ribosomes are obtained. Very recent structures of the yeast translocation factor and its complex with the antifungal drug sordarin reveal an unexpected conformational flexibility that might be crucial to the mechanism of translocation.