Protein Synthesis in Cell-Free Systems: an Effect of Interferon

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.     

Inhibition by soluble ribonucleic acid of stimulatory effect of liver template ribonucleic acid

1. Liver soluble RNA (s-RNA) and, to a smaller extent, Escherichia coli s-RNA inhibited the stimulation of [(14)C]leucine incorporation into protein in an E. coli S-30 system brought about by liver microsomal RNA or polyuridylic acid as template. 2. The inhibitory activity was associated with the fraction of the s-RNA possessing transfer-RNA activity. 3. The inhibition was exercised at a stage after charging of the s-RNA with amino acid. 4. Neither the method of preparation of the s-RNA nor its state of amino acid acylation affected its inhibitory action. 5. Stimulation of [(14)C]phenylalanine incorporation by polyuridylic acid or by liver microsomal RNA was not inhibited by addition of s-RNA. 6. It appears that excess of s-RNA inhibits the ambiguous incorporation of leucine with polyuridylic acid and also that something similar occurs with a natural template. 7. Estimation of messenger activity of samples of RNA should be carried out only after removal of s-RNA.     

Mechanism of Interferon Action: Inhibition of Viral Messenger Ribonucleic Acid Translation in L-Cell Extracts

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.     

The effect of age on protein synthesis in mouse liver

1. A system of microsomes and 105000g supernatant from livers of old mice is less able to promote the incorporation of [(14)C]phenylalanine into protein than a similar system from livers of young animals. 2. The decrease in [(14)C]phenylalanine incorporation is attributable to changes in microsomes from old animals rather than in the cell-sap fraction. 3. Decreased synthetic ability is found in various classes of microsomes from older animals, namely unfractionated, light and heavy microsomes, but not in detergent-washed ribonucleoprotein particles. 4. Deletions of certain detergent-soluble microsomal proteins accompany the decreased synthetic ability of microsomes from older animals. 5. Microsomes from old mice are less responsive to a synthetic messenger RNA, polyuridylic acid, and this is partly due to a higher rate of hydrolysis in the presence of cell sap from animals of extreme age. 6. Other more direct evidence, from the priming of a cell-free protein-synthesizing system from bacteria and the examination of ribonucleoprotein particles on sucrose density gradients, suggests that senescence is accompanied by a decrease in messenger RNA content.     

Partial Purification and Characterization of Messenger RNA Coding 14-3-2 Protein from Rat Brain

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.     

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.     

Inhibitory Effect of Diphtheria Toxin on Amino Acid Incorporation in Escherichia coli Cell-Free System

The mechanism of action of diphtheria toxin in an Escherichia coli cell-free protein-synthesizing system was examined. When the washed ribosomes were incubated with toxin before addition of messenger ribonucleic acid (RNA), peptide syntheses of (14)C-phenylalanine directed by polyuridylic acid or phage R17 RNA were strongly inhibited by a small amount of toxin. Whereas, if the soluble fraction (105,000 x g supernatant fraction) was preincubated with toxin, no effect of toxin occurred either on the induced protein synthesis or on the activity of guanosine triphosphatase even in the presence of nicotinamide adenine dinucleotide. The binding of (3)H-formylmethionyl-transfer RNA to E. coli ribosomes directed by either R17 RNA or trinucleotide AUG was also decreased by toxin. These findings suggest that diphtheria toxin may prevent the binding of messenger RNA by successfully competing with the AUG for ribosomal binding sites. Sucrose-density gradient studies support this concept by showing the decrease in binding of (3)H-labeled R17 RNA to E. coli ribosomes exposed to toxin.     

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.     

Protein Synthesis by a Cell-Free Preparation from the Bird Malaria,Plasmodium lophurae*

SYNOPSIS. 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 μg/ml heparin or 50 μg/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.     

Cell-free protein synthesis in a microfabricated reactor

Microbiochemical reactors having two inlet ports and one outlet port were fabricated on a silicon wafer by means of anisotropic etching in order to develop a parallel and automatic experimental system for cell-free translation. Using cell-free extract prepared from Escherichia coli, we tested the reactor for the translation of polyuridylic acid and MS2 phage RNA, and found that polypeptide and protein syntheses could be proceeded according to the genetic codes on the mRNAs. It indicates that the microfabricated reactor is useful for enzymatic reactions including complicated ones like cell-free translation. We also discuss the possibility of microsystems as advanced experimental tools for not only cell-free translation but also other various biochemical and biological research fields.     

Factors controlling amino acid incorporation by ribosomes from Krebs II mouse ascites-tumour cells

1. A ribosome-cell sap system capable of supporting the incorporation of (14)C-labelled amino acids into protein has been prepared from Krebs II mouse ascites-tumour cells. The requirements of this system for optimum activity and response to added messenger RNA have been investigated. One such system has been obtained for which amino acid incorporation is almost wholly dependent on the addition of suitable messenger RNA. 2. Ribosomes of widely different but predictable activities in the cell-free system have been prepared from Krebs cells pretreated in a variety of ways. The factors in the pretreatment of the cells responsible for these differences have been investigated. 3. The structural and functional properties of these different ribosome preparations and their response to exogenous messenger RNA have been examined and are discussed in the light of modern concepts of the control of protein synthesis.     

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 effects of polyuridylic acid on phenylalanine incorporation by subcellular fractions from carbon tetrachloride-poisoned rat liver

1. Ribosomes and microsomes isolated from the livers of rats that had received carbon tetrachloride 1hr. previously had decreased endogenous capacity to incorporate amino acid. 2. The capacity of the isolated structures to respond to a synthetic messenger, polyuridylic acid, and to incorporate phenylalanine was investigated. 3. It was found that ribosomes from carbon tetrachloride-treated animals, prepared with detergent and at high ionic strength, could be restored to the same specific activity as control particles with polyuridylic acid but that these particles required more Mg(2+) in the incubation mixture. 4. Microsomes could also be stimulated to control activities with polyuridylic acid, but had a narrow optimum range of Mg(2+) concentration. 5. Microsomes prepared from poisoned animals could be preprogrammed with polyuridylic acid to a significantly greater degree than could control particles, and this response was greater with increasing Mg(2+) concentrations. These data suggested that in carbon tetrachloride poisoning the messenger-ribosome interaction had been altered. 6. Attempts to deprogramme particles from control and treated animals resulted in decreased endogenous activity of both particles and a decreased capacity for the treated particles to be restored with the synthetic messenger. 7. It is suggested that two effects are present in carbon tetrachloride poisoning, namely an alteration of the messenger-ribosome interaction and an increased lability of the ribosome, as either separate or related events.     

Purification and analysis of murine 2-5A-dependent RNase

2-5A-dependent RNase (RNase L, RNase F) is an enzyme which mediates effects of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) in cells. 2-5A binding activity present in mouse liver extracts was measured using a 32P-labeled 2-5A derivative. Analysis of Scatchard plots was consistent with a single noninteracting 2-5A binding site with a Ka of 2.5 X 10(10) M-1. Similarly, affinity labeling of proteins with a 32P-labeled 2-5A derivative revealed a single, high-affinity 2-5A-binding protein of Mr 80,000. This 2-5A-binding protein was the only mouse liver protein specifically and consistently eluted by 2-5A from an affinity resin consisting of core(2-5A) covalently attached to cellulose. The 2-5A-eluted protein could degrade polyuridylic acid but not polycytidylic acid. Furthermore, when the 2-5A-eluted protein was electrophoresed into a polyuridylic acid-containing, nondenaturing gel, a band of degraded polyuridylic acid was demonstrated after incubation with 2-5A. There was no band of degraded polyuridylic acid when the elution was performed either in the absence of oligonucleotide or in the presence of low amounts of a closely related analog of 2-5A, p3I2'pA2'pA. Therefore, the Mr 80,000 2-5A-binding protein and the 2-5A-dependent RNase were almost certainly the same protein. Finally, the Mr 80,000 2-5A-binding protein was purified to homogeneity by electroelution from a polyacrylamide gel.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Cell-free translation of exogenous mRNA in extracts from dry pea primary axes

Extracts from the primary axes of dry pea (Pisum sativum L.) seeds are able to perform an initiation-dependent translation of exogenous mRNA. SDS polyacrylamide gel electrophoresis of the products synthesized under direction of alfalfa mosaic virus RNA (AMV-RNA) and tobacco mosaic virus RNA (TMV-RNA) shows that the fidelity of translation in this pea system is at least as high as in a wheat embryo cell-free protein synthesizing system. The endogenous messengers are also efficiently translated in extracts from the primary axes of pea seeds. The direct translation of these messengers in a homologous cell-free system may be of interest for a study of the products coded for by the long-lived messengers present in this plant.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulphate - mRNP messenger ribonucleoprotein - AMV-RNA alfalfa mosaic virus RNA - TMV-RNA tobacco mosaic virus RNA - ATA aurin tricarboxylic acid - TCA trichloroacetic acid - S.A. specific activity     

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.     

Modification of E. coli ribosomes and coliphage MS2 RNA by bisulfite: effects on ribosomal binding and protein synthesis.

The reaction of E. coli 70s ribosomes with 0.2 M NaH-35 s03 (pH 7.1, 3.5hrs, 37 degree) led to the conversion of 4.5% of the uracil residues of the R, RNA into 5.6-dihydrouracil-6-sulfonate residues. The modified ribosomes exhibited a significant decrease in their ability to bind (14-C)-phenylalanyl-(RNA-phe and to incorporate (14-C)-phenylalanine into protein in the presence of polyuridylic acid. The ability of the modified ribosomes to form an initiation complex as measured by the A-U-G or coliphage MS2 RNA dependent binding of (14-C)-fmet-tRNA-fmet was also impaired, as was their ability to incorporate (14-C) lysine into protein with MS2 RNA as messenger. Treatment os MS RNA with 0.2 M sodium (35-S) bisulfite, pH 7.0 at 25 degrees C resulted in the substitution of 2.7% and 6.2% of the uracil residues by bisulfite after 1 and 3.5 hrs of reaction, respectively. Impairment of function of the MS2 RNA in both initiation complex formation and transplantation assays was observed. These reactions of uracil residues of mRNA and rRNA may be a cause of biological damage inflicted by sodium bisulfite and sulfur dioxide.     

Ribonucleic acid synthesis during the early action of thyroid hormones

1. The effect on RNA synthesis in rat liver of thyroidectomy and the administration of thyroid hormone, especially during its physiological latent period, was studied by determining: (a) the activity of DNA-dependent RNA polymerase in isolated nuclei; (b) the rate of synthesis of nuclear and cytoplasmic RNA in vivo; (c) polyribosomal sedimentation profiles; (d) the response of microsomes and ribonucleoprotein particles to polyuridylic acid; (e) the effect of inhibitors of RNA and protein synthesis on the biological activity of hormones. 2. The DNA-dependent RNA-polymerase activity of isolated rat-liver nuclei was lowered by thyroidectomy and stimulated by the administration of tri-iodo-l-thyronine or l-thyroxine (2-25mug./100g. body wt.) to both normal and thyroidectomized rats. In thyroidectomized rats, the activity of the Mg(2+)-activated RNA-polymerase reaction (for which the product is mainly ribosomal type of RNA) was stimulated at 10-12hr. after a single injection of tri-iodothyronine, reaching a peak value of 60-90% stimulation at 45hr. after hormone administration. The Mn(2+)/ammonium sulphate-activated RNA-polymerase reaction (for which the RNA product is more DNA-like) was not affected for 24hr. after hormone administration but stimulated by 30-40% at 45hr. The response of both RNA-polymerase reactions to the hormone in vivo paralleled the physiological response but the enzyme was not stimulated by the addition in vitro of the hormone to isolated nuclei. 3. Within 3-4hr. after tri-iodothyronine administration to thyroidectomized rats, the specific activity of rapidly labelled nuclear RNA, after a 10min. pulse of [6-(14)C]orotic acid, was 30-40% greater than the control values, the stimulation reaching 100 and 200% at 11 and 16hr. respectively after hormone administration. Longer exposures to [6-(14)C]orotic acid and [(32)P]phosphate showed that the hormone accelerated the synthesis of mitochondrial, microsomal (or ribosomal) and soluble RNA. The greater part of the labelled nuclear RNA was of the ribosomal type. The hormone-induced increases in the incorporation of radioactive precursors into RNA were not preceded, but followed, by enhanced uptake of the precursor. There was no change, per g. of liver, of DNA, nuclear RNA or soluble RNA, but there was a 40-60% increase in the amount of ribosomal RNA between 35 and 45hr. after a single injection of tri-iodothyronine to thyroidectomized rats. 4. Coinciding with the increase in ribosomal RNA after hormone administration was an increase in the average size and amount of polyribosomes. The newly formed ribonucleoprotein particles, or messenger RNA attached to them, or both, were more firmly bound to microsomal membranes after hormone treatment. 5. Polyuridylic acid caused a bigger stimulation of incorporation of [(14)C]phenyl-alanine by ribonucleoprotein particles, but not by microsomes, from thyroidectomized rats as compared with preparations from normal animals. The response of ribonucleoprotein particles to polyuridylic acid was lowered after tri-iodothyronine treatment of thyroidectomized rats. 6. Actinomycin D, 5-fluorouracil, puromycin and cycloheximide caused a 70-100% inhibition of the stimulatory effect of l-thyroxine and tri-iodo-l-thyronine on basal metabolic rate and growth rate in both normal and thyroidectomized animals. Administration of actinomycin D also abolished the stimulation of RNA polymerase by tri-iodothyronine. 7. It is concluded that regulation of nuclear and ribosomal RNA synthesis is an essential step leading to the biological action of thyroid hormones and that the formation of new ribosomes is an important aspect of the control of cytoplasmic protein synthesis by these hormones.     

Internal initiation of polyuridylic acid translation in bacterial cell-free system

The task of the present work was to answer the question: is the free 5′-end needed for effective translation of a model polyribonucleotide template — polyuridylic acid — in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5′-end and the polyuridylic acid with blocked 5′-end were compared in the bacterial cell-free translation system. To block the 5′-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5′-end and uridylic oligoribonucleotide sequence at its 3′-end, schematically described as FAM(dC)₁₀(rU)₅₀, was covalently attached (ligated) to the 5′-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5′-blocked template and on the polyuridylic acid with free 5′-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5′-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.