Eukaryotic Cell-free Systems Research Articles

Translation of poly(A) in eukaryotic cell-free systems.

Translation of poly(A) in eukaryotic cell-free systems was investigated in comparison with that of poly(U), using the E. coli cell-free system as a control, and the following results were obtained. 1. Poly(A) was not translated in the eukaryotic cell-free systems prepared from yeast, wheat embryos, and rat liver, whereas poly(U) was well translated in these systems. On the other hand, fairly good translation of poly(A) as well as poly(U) was observed in the E. coli cell-free system. 2. The inefficient translation of poly(A) observed in the eukaryotic systems was not due to the lack of lysyl-tRNA synthetase, because of addition of [14C]lysyl-tRNA did not improve the synthesis of polylysine dependent on poly(A). 3. Eukaryotic ribosomes such as those prepared from Artemia salina did not bind [3H]poly(A) but bound [3H]poly(U) in the absence of tRNA, whereas they bound both polynucleotides if tRNA was present. Furthermore, the binding of [3H]poly(A) to A. salina ribosomes observed in the presence of tRNA was strongly dependent on the incubation temperature, indicating that the interaction between ribosomes and poly(A) was hampered by the base stacking known to be present in poly(A). Thus, tRNA seemed to be required for the formation of the stable ribosome-poly(A) complex. 4. Binding of [14C]lysyl-tRNA to ribosomes, either of A. salina or of E. coli, was observed as well as that of [14C]phenylalanyl-tRNA. In contrast, chain elongation of polylysine was extremely slow in the eukaryotic system as compared with the E. coli system, although chain elongation of polyphenylalanine proceeded equally well in both systems, and this resulted in poor synthesis of polylysine in the eukaryotic systems.

Preparation of a mRNA-dependent cell-free translation system from whole cells of Saccharomyces cerevisiae.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by mechanical breakage of cells, isolation of a 30000 x g supernatant fraction and removal of endogenous mRNA by treatment with micrococcal nuclease. The system thus isolated is dependent on added mRNA and translates yeast mRNA to discrete products, many of then identical with yeast proteins synthesized in vivo. Activity and properties of this system are comparable to those of other eukaryotic cell-free translation systems. It offers the following advantages, compared to yeast translation systems described previously. (a) Its isolation is simple and fast. (b) Since it is not isolated from spheroplasts there is no danger of its inactivation by contaminants in enzymes used for spheroplast preparation. (c) Isolation appears to be less strain-dependent and can be carried out starting from cells in various physiological states.

The effects of beta-endorphin and enkephalins on protein biosynthesis in a eukaryotic cell-free system. Inhibition of phenylalanyl-tRNA synthetase.

The effects of beta-endorphin and various enkephalins on protein synthesis in eukaryotic cell-free systems have been examined. Beta-Endorphin, Leu-enkephalin, and Met-enkephalin inhibit the incorporation of radioactive leucine into globin in the presence of reticulocyte poly(A)+ RNA and of radioactive phenylalanine into polyphenylalanine in the presence of poly(U); however, the poly(U)-dependent synthesis of polyphenylalanine from Phe-tRNA is not inhibited. the aminoacylation of tRNAPhe is markedly inhibited by enkephalin, indicating that the sensitive component is Phe-tRNA synthetase. Other aminoacyl-tRNA synthetases are not significantly affected. The interaction between enkephalin and Phe-tRNA synthetase is reversible; activity is restored by dialysis of enzyme-enkephalin reaction mixtures. Morphine, vasopressin and analogues of vasopressin, and enkephalin analogues such as [D-Ala2,D-Leu5]enkephalin and [D-Ala2,Met]enkephalinamide have no effect on translation. The results suggest that the effects on protein synthesis are probably not related to opiate effects.

Action of membrane-active compounds on mammalian cells. Permeabilization of human cells by ionophores to inhibitors of translation and transcription.

1. Hygromycin B, an inhibitor of protein synthesis in eukaryotic cell-free systems, does not block translation in intact HeLa cells, as mammalian cells are normally impermeable to this antibiotic. However, the presence in the culture medium of the ionophore nigericin at concentrations that did not interphere with protein synthesis rendered human HeLa cells permeable to hygromycin B. A similar effect was achieved with a number of ionophores, such as valinomycin, monensin, gramicidin D, bromolasalocid, A23187, amphotericin B and nystatin, as well as with the cardioglycoside antibiotic ouabain, which blocks (Na+, K+)-ATPase activity. The permeabilization of HeLa cells by nigericin to several inhibitors of translation was also studied. The inhibition of protein synthesis by destomycin A, anthelmycin, gougerotin, edeine, tetracycline and several nucleotide derivatives in cultured HeLa cells was greatly enhanced by the presence of 0.8 μM nigericin. 2. Concentrations of α-amanitin above 200 μ/ml were required to inhibit RNA synthesis completely in intact cells. Once more, the presence of nigericin reduced 20–50-fold the concentration of α-amanitin necessary to inhibit transcription. Interestingly enough, nigericin alone proved to be a very effective inhibitor of protein synthesis in HeLa cells: 2 μM nigericin almost completely abolished the translation capacity of the cells, whereas transcription was much more resistant to inhibition by nigericin. 3. The concentration of monovalent cations in the culture medium drastically affected: (a) the permeability of HeLa cells to hygromycin B, (b) the inhibition of protein synthesis by nigericin and (c) the permeability of HeLa cells to hygromycin B induced by nigericin. A very simple method of permeabilizing cultured cells to low-molecular-weight compounds has been devised. This method, which permits translation in HeLa cells to continue at control levels under the permeabilization conditions, is very mild and is based on the manipulation of the monovalent ion concentrations of the culture medium.

Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure

In this and the accompanying paper we demonstrate that certain prokaryotic mRNAs, when modified at their 5'-termini with a cap structure, are translated in a eukaryotic cell-free protein synthesising system as efficiently as, or more efficiently than, eukaryotic mRNAs. Apparently, the prokaryotic mRNA contains all the information necessary for efficient recognition and initiation by eukaryotic translational components, except for the cap structure.

Efficient cap-dependent translation of polycistronic prokaryotic mRNAs is restricted to the first gene in the operon

Certain polycistronic prokaryotic mRNAs, when modified at their 5'-termini with a cap structure, are translated as efficiently as, or more efficiently than eukaryotic mRNAs in a eukaryotic cell-free protein synthesising system. However, in this case efficient cap-dependent translation is apparently restricted to the 5'-proximal coding sequence. Moreover, certain translational regulatory signals potentially used by these prokaryotic mRNAs to regulate their levels of expression seem to be recognised by the eukaryotic translational components. The evolutionary significance and practical implications of these results are discussed.

Inhibition of protein synthesis by Cl-.

Optimum K+ concentration for protein synthesis in four eukaryotic cell-free systems is obtained with 70 to 80 mM added KCl or with 110 to 150 mM added K(OAc). The different K+ optima are due to inhibition of protein synthesis by Cl- at concentrations higher than those present in the cytoplasm of eukaryotic cells. Initiation of protein synthesis is severely inhibited with 150 mM added KCl. This inhibition results from an impairment of mRNA binding to ribosomes. The binding of initiator Met-tRNAt, however, is only slightly inhibited by 150 mM KCl.

Bacteriophage T3 and T7 early RNAs are translated by eukaryotic 80S ribosomes: active phage T3 coded S-adenosylmethionine cleaving enzyme is synthesized.

RNA transcribed in vitro from the early region of bacteriophage T3 or T7 was translated by cytoplasmic ribosomes which synthesized protein in cell-free systems prepared from mammalian cells and wheat germ. The proteins synthesized in vitro and their counterparts prepared from infected Escherichia coli comigrate by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and are similarly affected by deletion or amber bacteriophage mutations. Bacteriophage T3 codes for an enzyme that cleaves S-adenosylmethionine and this activity was detected among the products of the mammalian cell-free system. Bacteriophage T3 or T7 RNA, after endoribonuclease III (EC 3.1.4.24) cleavage, gave higher levels of incorporation into phage T3 or T7 polypeptides than when an equivalent amount of the uncleaved RNA was added to the eukaryotic cell-free systems. Methylation of phage T3 or T7 RNAs is apparently not required for translation in either the wheat germ or mammalian cell-free system. The ability of T3 and T7 RNA to be translated in the presence of saturating amounts of natural eukaryotic mRNAs suggests that many prokaryotic genes introduced into mammalian cells might be expressed if they were transcribed in an appropriate form.

Translation of murine leukemia virus RNA in cell-free systems from animal cells.

The virion RNA of Moloney murine leukemia virus (MuLV) has been translated in eukaryotic cell-free systems derived from mouse L- and human HeLa cells. In both systems at least three polypeptides, approximately 60,000, 70,000, and 180,000 in apparent molecular weight, were formed in response to the added 35S MuLV RNA. All three polypeptides were precipitable with antiserum to detergent-disrupted MuLV. Fingerprint analysis of tryptic digests indicated that all three contain anino acid sequences in common with each other and with the major methionine-containing structural proteins of the virion.

Translation of a specific mRNA from [formula omitted] in a eukaryotic cell-free system

A specific mRNA for a structural lipoprotein of the Escherichia coli outer membrane was translated in a wheat germ cell-free protein synthesizing system, S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) had neither stimulative nor inhibitory effect on the translation. When the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two peaks appeared at the appearent molecular weights of about 15,000 and about 7,500. Both products were cross-reactive with antiserum against the lipoprotein.

Complete translation of poliovirus RNA in a eukaryotic cell-free system.

Poliovirus RNA stimulates imcorporation of 35S from both [35S]methionine and formyl-[35S]methionyl-tRNAfMet in cell-free systems derived from HeLa cells or from poliovirus-infected HeLa cells. The largest product formed under the direction of the viral RNA is the same size as the polyprotein thought to represent translation of the entire RNA. Synthesis of this polyprotein and other large products was stimulated greatly by increasing the salt concentration during the reaction from the optimum for initiation (90 mM) to the optimum for elongation (155 mM). Only one initiation peptide could be identified, and a tryptic digest of the product contained mainly peptides that cochromatographed with peptides from authentic viral proteins. The RNA from a deletion mutant of poliovirus initiated protein synthesis at the same site used by standard RNA and programmed synthesis of an appropriately deleted set of polypeptides. The results strongly support the model of translation of poliovirus RNA from a single initiation site into a continuous polyprotein that is cleaved to form the functional proteins. It is suggested that uninfected HeLa cell extracts can carry out the cleavages of nascent polyprotein.