Characterization of a protein synthesis system from rat liver. Translation of endogenous and exogenous messenger RNA

An in vitro system prepared from rat liver postmitochondrial supernatant exhibits a high rate of protein synthesis for an extended period of time. This system initiates translation of either endogenous or exogenous mRNA, incorporates Met at a rate of 13 pmol/mg of postmitochondrial supernatant protein/min, maintains this rate for at least 90 min, and performs several rounds of translation/mRNA molecule. Up to 50% of the activity is due to reinitiation of protein synthesis using endogenous mRNA. In addition, 60-70% of the protein synthesized was released from ribosomes into the medium. Addition of globin mRNA stimulates protein synthesis and results in the synthesis of a protein that comigrates with authentic rabbit globin. Black beetle virus mRNA 2 also stimulates protein synthesis and results in synthesis of a protein with molecular weight corresponding to that of the mature viral protein. With endogenous rat liver mRNA this system synthesizes a large number of proteins.     

Evidence for the presence of mRNA in the post-ribosomal cytoplasm of sheep lymphocytes.

Several fractions of RNA prepared from the post-ribosomal cytosol of sheep lymphoid cells were found to include messenger-like RNA as defined by the following criteria: a, template activity, i.e. the ability to promote the incorporation of radioactive amino acids into protein in cell-free protein-synthesising systems derived from wheat embryos or ascites tumour cells; b, a low magnesium optimum (1-2.5 mM) for template activity which is characteristic of many natural mRNAs; c, sensitivity of the template response to aurintricarboxylic acid, a specific inhibitor of the initiation of protein synthesis. The lymphoid post-ribosomal RNA fractions, however, were translated less efficiently than were rabbit reticulocyte globin mRNA or tobacco mosaic viral (TMV) RNA; no explanation for this relatively poor template activity was found. The major fraction of messenger-like RNA had an average sedimentation coefficient of 12 S; this fraction directed the translation of several discrete polypeptides in the molecular weight range 10 000-25 000. On average the products of 12 S RNA-directed protein synthesis appeared lysine rich compared with TMV RNA-directed products. It is suggested that the apparent pool of uncommitted mRNA in resting lymphocytes may be utilised during the early stages of lymphocyte activation, and that the mRNAs could be stored in forms similar to those evident in other dormant tissues.     

Protein synthesis and aging: studies with cell-free mammalian systems.

A cell-free system devoid of polysomes, which translates natural mRNA, has been prepared from rat liver. It contains ribosomal subunits, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and protein factors necessary for translation. Protein synthesis required an energy-generating system, mRNA, and 3 mM Mg2+ concentration, and it was inhibited by 7-methylguanylic acid. The total extent and the rate of protein synthesis were approximately 30% greater when the translating system was prepared from livers of 3-month-old rats, as compared to 30-month-old rats. A ribosome-free fraction containing the protein factors required for translation was also prepared from 3-month-old and 30-month-old rat livers and brains, by extraction with 0.5 M KCl. The high-salt extracts were analyzed for elongation factors EF-1 and EF-2 in a poly(U) translating system. Although the activity of EF-2 was similar in preparations from young and old rats, the EF-1 activity in the 3-month-old rat livers and brains was 30 to 40% greater than in 30-month-old animals. The protein synthesizing activity of high salt-washed ribosomes stripped of endogenous peptidyl-tRNA and mRNA, from livers and brains of young and old animals, was the same.     

Cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mRNA''s in extracts from uninfected and infected Ehrlich ascites tumor cells.

The mechanism whereby picornaviruses inhibit host protein synthesis while their own synthetic processes proceed unabated has remained elusive. One of our approaches to this problem was to study the ability of cell-free extracts derived from uninfected and mengovirus-infected Ehrlich ascites tumor cells to translate viral and nonviral mRNA's under various conditions of incubation. Our results indicate that viral messengers (from mengovirus and encephalomyocarditis virus) and cellular messengers [L cell and Ehrlich ascites tumor poly(A)-containing mRNA's, rabbit globin mRNA, and chicken embryo lens crystallin mRNA] are translated equally well in both extracts. We also examined the simultaneous translation of viral and nonviral mRNA's in extracts from uninfected Ehrlich ascites tumor cells. Our results indicate that under certain conditions mengovirus RNA can suppress completely the translation of globin mRNA. The significance of these results in terms of the shutoff of host protein synthesis is discussed.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Evidence for simultaneous derepression of messenger RNA and the guanine nucleotide exchange factor in fertilized sea urchin eggs

Translational control was studied in extracts of Lytechinus pictus eggs and zygotes. We showed that neither mRNA nor initiation factors alone limit translation in these lysates; rather they are together rate limiting. Added globin mRNA was translated in egg and zygote lysates but overall protein synthesis did not increase significantly as the added RNA competed with the endogenous message. The lysates mimicked the in vivo response, since microinjection of globin mRNA into L. pictus eggs similarly competed with endogenous mRNAs. A number of translational components were used to determine if they would stimulate protein synthesis in these lysates. The addition of globin polyribosomes increased the level of protein synthesis. The majority of this increase was due to reinitiation of the globin mRNA, and under these conditions the level of endogenous protein synthesis in both egg and zygote extracts did not change. The addition of crude initiation factors alone did not appreciably alter the rate of protein synthesis in the egg lysates. However, in the presence of added mRNA, these initiation factors stimulated translation two- to fourfold. Of all the initiation factors tested, only the guanine nucleotide exchange factor (GEF, eIF-2B, RF) significantly increased protein synthesis when globin mRNA was present. The addition of an unfractionated initiation factor preparation further stimulated protein synthesis in the presence of added GEF and mRNA, suggesting that a component other than mRNA and GEF was also limiting in these egg lysates. Other initiation factors, including eIF-2, eIF-4A, eIF-4B, and eIF-4F, did not substitute for the component in the unfractionated initiation factor preparation. We propose that alkalinization of the cytoplasm and the subsequent activation of initiation factors and mRNAs contribute to the large stimulation of protein synthesis in echinoid eggs after fertilization. Furthermore, we discuss the possibility that the increase in NADPH at the expense of NAD+, which occurs within 3 min after fertilization, may lead to the activation of GEF.     

Preparation and characterization of cell-free protein synthesis systems from oocytes and eggs of Xenopus laevis

During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells. The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.     

Synthesis of rabbit globin in a cell-free protein synthesis system utilizing sea urchin egg and zygote ribosomes

Reports of the reduced ability of sea urchin egg ribosomes to participate in synthetic mRNA-directed protein synthesis have fostered the suggestion that the low protein synthesis rate of eggs is due to ribosome-associated inhibitors. To test this hypothesis with a natural message, we have isolated 80S ribosomes and microsomal ribosomes of sea urchin eggs and zygotes and compared their activity at synthesizing protein from rabbit α and β globin mRNA in a Krebs II ascites tumor cell-free system. Both egg and zygote 80S ribosomes responded to added mRNA and were shown to synthesize complete α and β globin chains by CM-cellulose chromatography. In most cases, the activity of the egg ribosomes was in comparable instances higher than the zygote ribosomes. Attempts to determine the cause of this difference indicated that it was not a function of K⁺ or Mg²⁺ concentration, type of tRNA used, or ribosomal wash proteins. From these studies it is apparent that sea urchin egg ribosomes are functional at a level equivalent to or better than zygote ribosomes, and it appears that the lack of protein synthetic activity in unfertilized eggs is not due to the presence of a population of inhibited ribosomes.     

Defective initiation on natural messenger RNA by cell free systems from Krebs ascites cells incubated at elevated temperatures.

1. Cell-free systems prepared from Krebs II ascites cells incubated at 45 degrees C have a much lower endogenous activity than those from cells incubated at 37 degrees C. The endogenous activity is mainly due to completion of polypeptide chains initiated in the intact cell. The low activity of the 45 degrees C system is due to a lesion in initiation in cells incubated at 45 degrees C. 2. Cell-free systems from cells incubated at 45 degrees C can translate efficiently poly (U) at 8 mM Mg2+. However, they initiate poorly on globin mRNA, indicating that these systems reflect the situation in the intact cell. 3. The lesion in globin mRNA translation in 45 degrees C systems can be overcome by addition of reticulocyte initiation factors. At saturation concentrations of factors, the response of a 45 degrees C system is restored to almost normal. 4. 45 degrees C systems from 40-S initiation complexes with methionyl tRNAfmet almost as efficiently as normal, but their ability for form 80-S complexes with globin mRNA is impaired, unless they are supplied with exogenous initiation factors.     

Regulation of protein synthesis in mitotic HeLa cells.

Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mol wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.     

Inhibitory action of different RNA fractions on the translation of globin mRNA in vitro.

Preparations of 28S rRNA and 12S RNA, obtained from sheep lymphocytes, were shown to inhibit the translation of globin mRNA. An inhibition by a given amount of 12S or 28S RNA was only obvious at saturating or near saturating levels of globin mRNA, suggesting that the inhibitory RNAs interacted with some factor essential for protein synthesis other than mRNA. The inhibitory RNAs had no effect on the translation of the synthetic template polyuridylic acid. It is suggested therefore that the target for inhibitory RNAs might be a natural mRNA specific initiation factor.     

A cell free system from HeLa cells active in initiation of protein synthesis.

A cell free system programmed by endogenous mRNA and active in initiation of protein synthesis has been obtained from HeLa cells by adding 25-100 muM hemin to the medium used to homogenize the cells. Hemin stabilizes the initiation activity of the extract, which otherwise decays rapidly even at 0 degrees C. The role of hemin in promoting initiation has been examined by fractionating the extracts into ribosomes and postribosomal supernatant (S150). An extract prepared without hemin or the S150 obtained from this extract prepared without hemin or the S150 obtained from this extract inhibits protein synthesis of the extract containing hemin by about 30%. The ribosomes prepared from extracts containing hemin are active in initiation of protein synthesis, whereas the ribosomes obtained from the extracts prepared without hemin show little or no initiation. These results have suggested that addition of hemin prevents the formation of an inhibitor of initiation in the S150 and at the same time protects from inactivation an initiation factor associated with ribosomes or ribosomal subunits. Addition of 2 mM GTP to HeLa extracts stabilizes the initiation activity, though to a smaller degree than hemin. The effects of hemin and GTP are not additive, suggesting that they may act on the same target molecule, though possibly by different mechanisms. The mechanism of action of GTP is discussed in view of similar observations made in the rabbit reticulocyte cell free system.     

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

The rate of initiation of alpha- and beta-globin mRNA translation is modulated by 50 kDa, 28-kDa and 24-kDa polypeptide-containing fractions

The relative rates of initiation of alpha- and beta-globin mRNA translation in a Krebs II ascites cell-free system are differently modulated by a 50-kDa protein and two fractions containing either a 28-kDa or a 24-kDa polypeptide. Each of these fractions stimulated a discrete step that limits initiation of protein synthesis, but other rate-limiting steps take place upstream and/or downstream, resulting in characteristic kinetics of the stimulation of alpha- and beta-globin synthesis. The ascites extracts appear to be deficient in these activities.     

A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.     

Protein synthesis and ribosome activation during the early stages of phytohemagglutinin lymphocyte stimulation

The rate of protein synthesis by lymphocytes increases linearly from 3 h after the addition of phytohemagglutinin (PHA). There is a linear increase in the percentage of active ribosomes between 3 and 12 h of lymphocyte culture with PHA. Thus, an important early event during the activation of lymphocytes by mitogen is a stimulation in the rate of initiation of protein synthesis.     

Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems

A highly efficient mammalian system was developed for the in vitro translation of exogenous rabbit globin messenger RNA. The system consists of purified ribosome subunits from mouse liver, rabbit reticulocytes, or guinea pig brain, partially purified initiation factors from rabbit reticulocytes, and elongation factors, termination factors, aminoacyl-tRNA synthethases and tRNA from rat liver in the form of pH 5-enzymes. (1) Emphasis was put on well-defined, structurally and functionally intact ribosomes, which we found to be the most difficult component of the system in terms of stability of activity. (2) An improved method for extraction of initiation factors from crude reticulocyte ribosomes was developed. Factor preparations of high specific activity were obtained by a simple, partial purification procedure. The crucial point was not to damage the ribosome structure during extraction of the initiation factors and to eliminate inhibitory components during extraction and purification. (3) The efficiency of the system was demonstrated quantitatively by showing that between $\text{3}\text{4}$ and one mRNA molecule per ribosome saturates the system and that each ribosome recycles over the mRNA several times. (4) Major uncertainties and ambiguities in the search for and identification of true initiation factors, as opposed to structural ribosome proteins needed for the reconstitution of damaged ribosomes, can be reduced by using this system.     

Messenger RNA affinity column fractionation of eukaryotic initiation factor and the translation of myosin messenger RNA.

Both myosin mRNA (26 S) and globin mRNA (9 S) have been bound to activated Sepharose 4B. The affinity of initiation factors derived from native 40 S ribosomal subunits from embryonic chick muscle for these messengers has been determined. Although both messengers bind the major components of the muscle factor preparation with the same affinity, some differences are noted in the minor components. There is an enrichment of components which bind myosin mRNA with a high affinity when the 15–18 S initiation factor complex is prepared from initiating 40 S ribosomal subunits found on myosin synthesizing polysomes rather than from total cellular factor preparations. The proteins which have a high binding affinity to myosin mRNA also have a discriminating effect when added to a wheat germ system containing myosin and globin mRNA. This is demonstrated by the fact that the synthesis of myosin heavy chain is specifically stimulated and the number of ribosomes found on myosin mRNA increase five to seven-fold; whereas neither the synthesis of globin nor the number of ribosomes associated with globin mRNA is increased. The components of an impure reticulocyte eukaryotic initiation factor 3 prepared in a similar manner as the muscle factor, do not bind myosin mRNA with the same high affinity, and these fractions separated on the myosin mRNA affinity column did not show a discriminatory effect. These results suggest that specific components of muscle 15–18 S initiation factor preparations have a higher binding affinity for myosin mRNA than globin mRNA and that these proteins may be those factors previously reported to be present which discriminate between mRNAs.     

Mechanism of inhibition of hepatic protein synthesis in rats by the carcinogen, methylazoxymethanol acetate.

Treatment of rats with the carcinogen, methylazoxymethanol acetate, results in a rapid, marked inhibition of hepatic protein synthesis and disaggregation of polysomes. Studies were undertaken to learn the mechanism by which this carcinogen induces these effects in rat liver. The data show that the inhibition of endogenous protein synthesis is not due to an effect on the high speed supernatant 'factors' but rather at the level of the polysome, and that both free and membrane-bound polysomes are affected. Poly(U)-directed polyphenylalanine synthesis by native ribosomal subunits is greater in preparations isolated from rats treated with carcinogen than it is in controls. Moreover, the native ribosomal subunit fraction from treated livers in response to added rabbit globin mRNA is able to synthesize a protein similar in molecular weight to globin. These studies show that methylazoxymethanol acetate does not induce significant alterations of ribosomal subunits or of initiation factors and suggest that the inhibition of protein synthesis and disaggregation of polysomes may be the results of an alteration of cytoplasmic mRNA, or its association with ribosomes.