Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation.

It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCl, that the optimum concentration of potassium acetate is higher than for KCl, and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs. When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found. The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and was very much more efficient and accurate with KCl rather than potassium acetate. It was also unique in being strongly activated by low concentrations (5-10 mM) KSCN in the presence of 90 mM potassium acetate. For the translation of other uncapped RNAs (poliovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl. However, KCl was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the result that KCl gave a product pattern closer to that observed in vivo. In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KCl rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.     

Role of tissue specific factors in the translation of brain messenger ribonucleic acids in vitro

The activity of brain ribosomal subunits was examined by measuring (a) the saturation of ribosomes with nascent polypeptides and the rates of release of completed chains; (b) interaction of the subunits with brain messenger RNA to form polysomal aggregates; (c) formation of polyphenylalanine in the presence of polyuridylic acid at high magnesium concentration and (d) inhibition by aurine tricarboxylic acid. The results showed that a portion of the subunits were defective in forming initiation complexes with brain messenger RNA, but translated polyuridylate efficiently. The subunits that did form polysomes were more competent than the heterologous systems (derived from Krebs ascites cells, reticulocytes or wheat germ) in carrying out reinitiations of brain mRNA translation.Both the homologous and the heterologous systems translated brain mRNA and synthesized the two brain specific proteins S-100 and the neuron specific enolase, indicating that each of the systems had all the necessary factors. However, homologous initiation factors, aminoacyl-tRNA synthetases and transfer RNAs were more effective, particularly at suboptimal concentrations. Our results suggest that discriminative translation of brain messenger RNA may take place based on relative proportions of required components in the reaction milieu rather than by the presence or absence of one or more special messenger RNA specific factors.     

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.     

Differential effects on RNA translation by a KC1 extract of reticulocyte ribosomes: characteristics of an inhibitory fraction

A KCl extract of rabbit reticulocyte ribosomes has been demonstrated to markedly stimulate the translation of various messenger RNAs in a cell-free system from Krebs II ascites tumor cells. In contrast, the translation of encephalomyocarditis viral RNA is strongly inhibited by the same extract. Fractionation of the KCl extract allows the separation of these inhibitory and stimulatory activities. The inhibitory activity has been shown to be the consequence of an unusual endonuclease, associated with ribosomes, that produces approximately 4 S products from the degradation of globin mRNA and viral RNA.     

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.     

鲮鱼垂体Poly(A)~+RNA的分离和纯化及其生物活性鉴定

本文利用快速保温法分离纯化了鲮鱼垂体Poly(A)~+RNA,并首次在北农大“白粒146号”小麦麦胚体系中进行了体外翻译活性测定,对鲮鱼Poly(A)~+RNA其最适镁离子浓度为1.7mmol/L,最适Poly(A)~+RNA浓度为0.12μg/50mL,但钾离子浓度及预保温对蛋白质的合成影响不大。实验结果表明鲮鱼垂体Poly(A)~+RNA的体外蛋白翻译活性达对照组的22倍。由于改进了方法,简化了操作程序,因此使鲮鱼垂体Poly(A)~+RNA的翻译活性大大提高了。     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ.

The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated. As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA. Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli. The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis. Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64. Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Transport of ornithine carbamoyltransferase precursor into mitochondria. Stimulation by potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s)

The precursor of rat liver ornithine carbamoyltransferase (EC 2.1.3.3) synthesized in vitro was taken up and processed to the mature enzyme by isolated rat liver mitochondria. Potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s), in addition to the precursor and the mitochondria, were required for maximal transport and processing of the precursor. The concentrations of potassium and magnesium ions required for maximal transport and processing were about 120 and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36 mg of protein/ml), in combination with potassium and magnesium ions, stimulated the transport and processing severalfold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heating at 100 degrees C for 2 min. No significant amount of the precursor was associated with the mitochondria when incubation was performed in the absence of these components. These results suggest that potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s) stimulate the binding and transport of the ornithine carbamoyltransferase precursor to the mitochondria. Dialyzed supernatant of rabbit erythrocyte lysate was equally effective in stimulating the precursor transport and processing, and a dialyzed cytosol fraction of Ehrlich ascites cells was partly stimulatory. On the other hand, dialyzed cytosol fractions of rat liver and rat kidney, and dialyzed supernatant of wheat germ extracts did not stimulate the precursor transport and processing but rather inhibited it.     

Preparation and properties of an improved cell-free protein synthesis system from mammalian liver

A cell-free protein synthesis system derived from mouse liver has been developed which faithfully translates endogenous and exogenous mRNA. The system is based on the unfractionated post-mitochondrial supernatant. The main measures taken to improve the activity of the system were: the use of high levels (30 mM) of creatine phosphate as an energy-generating system to counteract a hyperactive nucleoside triphosphatase activity in the extracts, the choice of homgenisation buffer, and the use of potassium acetate rather than KCl in the assay. The system exhibits a high initial rate of amino acid incorporation, and reinitiates translation on endogenous mRNA. Added tobacco mosaic virus (TMV) RNA is faithfully translated into full-length products at a rate of 60-80 amino acid residues per min at 30 degrees. The rate of overall amino acid incorporation slows after about 20 min and eventually ceases due to a failure in the re-initiation of translation, and not because of degradation of mRNA. Over a limited period of time, this improved cell-free translation system is comparable in activity to other eukaryotic systems generated to date, and should be useful in studies of the control of translation rates in mammalian liver.     

Cell-free Synthesis of the Major Storage Protein of the Bean, Phaseolus vulgaris L

As seeds of the French bean (Phaseolus vulgaris, L. cv. Tendergreen) mature, a single protein, G1 globulin (analogous to legumin), represents the majority of protein synthesized. Washed polysomes extracted from developing cotyledons had little endogenous activity in amino acid incorporation, but on addition of cell-free extracts from wheat germ, active incorporation was obtained, the level being similar to that with viral RNA as messenger. The Mg(2+) optimum for protein synthesis in the presence of bean polysomes was 6 mm compared with 4 mm for synthesis of viral polypeptides in the wheat germ system. Using T-2 toxin as an inhibitor, it was shown that 29% of the incorporation depended on initiation events. Electrophoretic analysis of the total polypeptide products of cell-free synthesis gave a disperse profile. Centrifugation to remove polysome-bound peptides after 60 minutes incubation gave a supernatant having a product with the same electrophoretic mobility as G1 globulin and containing 26% of the radioactivity present in the gel. Protein eluted from this peak was subjected to re-electrophoresis and shown to consist of the three polypeptide subunits characteristic of G1 globulin.     

Variation of Cell-free Translation Profiles Among Pathogenic Strains of Soybean Mosaic Virus

Cell-free translation products from isolates representing soybean mosaic virus (SMV) strains G1 to G7 and G7a, along with several other SMV isolates, were analyzed. SMV RNAs were translated in both rabbit reticulocyte lysates and wheat germ extracts, yielding approximately 20 translation products for each strain from each translation system. Comparison of translation profiles by the presence or absence of proteins allowed for the formation of distinctive groups from each cell-free translation system. Groupings formed by analysis of products from rabbit reticulocyte lysates correlated with pathogenicity; groupings formed by analysis of products from wheat germ extracts had no apparent biological significance.     

双抗体免疫沉淀法纯化牛生长激素poly(A)~+RNA的研究

我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91％可以被牛生长激素抗体沉淀。用SDS-11％PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.     

Cell-free Synthesis of the Myelin Basic Proteins in a Wheat Germ System Programmed with Brain Messenger RNA

Poly A(+) messenger RNA (mRNA) was isolated from the brains of 3-week-old mice and translated in a cell-free system derived from wheat germ. Maximal stimulation of the system by brain mRNA was observed at a relatively low K+ concentration (45 mM) and low mRNA concentration (1-10 microgram/ml). The translational system was dependent on an energy-generating system and stimulated by the addition of spermidine and transfer RNA. Under optimal conditions, incorporation was linear for almost 45 min, but the overall stimulation with brain mRNA was relatively low (about twofold). In spite of the low stimulation, analysis of the translation products indicated that in the presence of brain mRNA polypeptides which co-chromatographed and co-electrophoresed with the two mouse myelin basic proteins could be detected. In control experiments with liver poly A(+) mRNA, which stimulated the translational system to a greater extent than brain mRNA, no such polypeptides could be detected. In this system the ratio of synthesis of small myelin basic protein to large myelin basic protein was found to be about 4.0, which correlates well with that found in vivo.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

Cell-free protein synthesis in lysates of Drosophila melanogaster cells.

A procedure is described for preparing cell-free protein synthesizing lysates from Drosophila melanogaster tissue culture cells and embryos. Preparation of translationally active lysates from tissue culture cells is dependent on the presence of rat liver supernatant during cell lysis to inhibit ribonuclease activity. After micrococcal nuclease treatment of the lysate, protein synthesis is dependent on the addition of exogenous messenger RNA. The fidelity of translation is very high. The conditions for optimal translation have been determined. In addition, the effects on translation of a variety of supplements, including rat liver supernatant, have been analyzed. The products of translation by the Drosophila lysate have been compared with those of wheat germ extracts and of micrococcal nuclease treated rabbit reticulocyte lysates. Translation in vitro of bovine parathyroid hormone messenger RNA yielded two products tentatively identified as preproparathyroid hormone and proparathyroid hormone, as well as an unidentified third product. This result suggests that insect enzymes can accurately process mammalian precursor proteins.     

Nonuniform size distribution of nascent peptides: The role of messenger RNA

The origin of the nonuniform size distribution of nascent rabbit globin peptides has been investigated in the reticulocyte lysate and wheat germ cell-free protein synthesis systems. Increasing the concentrations of the cellular components involved in protein synthesis failed to alter the elution pattern observed upon chromatographic analysis of reticulocyte lysate nascent chains. Nascent chains isolated from a globin messenger RNA-directed wheat germ cell-free system showed a nonuniform size distribution of nascent peptides similar to that of the rabbit reticulocyte nascent chains. These observations indicate that the nonuniformity of the globin nascent chains arises from a unique property of the messenger RNA being translated and not from limiting concentrations of a component or components of the reticulocyte protein synthesis system.