Mechanism of interferon action: inhibition of pppA(2'p5'A)n-dependent ribonucleases activity in micrococcal nuclease-treated mouse L cell-free extracts

The ability of nonpreincubated as compared to micrococcal nuclease-treated mouse L cell-free extracts supplemented with 2′-deoxythymidine-3′, 5′-diphosphate (pTp) and ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) to catalyze the 2′,5′-oligoadenylate-dependent degradation of reovirus [³H]mRNA was investigated. 2′,5′-Oligo A tetramer enhanced degradation in nonpreincubated but not micrococcal nuclease-treated extracts. Neither pTp nor EGTA significantly affected the 2′,5′-oligo A-dependent degradation in nonpreincubated extracts. The presence of both micrococcal nuclease and calcium was required to establish the subsequent reduction in both 2′,5′-oligo A-dependent and -independent degradation observed in micrococcal nuclease-treated extracts containing pTp and EGTA.     

Translational control by messenger RNA competition for eukaryotic initiation factor 2

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.     

Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus infected L cells

Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.     

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.     

Preferential stimulation of rabbit α globin mRNA translation by a cap-binding protein complex

A cap-binding protein complex (Edery et al. (1983) J. Biol. Chem. 258, 11398–11403) is shown here to stimulate preferentially the translation of endogenous α versus β globin mRNA in a rabbit reticulocyte lysate. Several initiation factors (eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4E and eIF-5) and elongation factor 1 were found to have no such discriminatory effect. These results are in contrast to several previous reports and demonstrate that the only factor capable of relieving translational competition between α and β globin mRNAs is the cap-binding protein complex.     

Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity.

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.     

A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.     

Increase in eukaryotic initiation factor 2B activity following fertilization reflects changes in redox potential.

One of the factors involved in the postfertilization activation of protein synthesis in the sea urchin, Strongylocentrotus purpuratus, is the activation of eIF-2B, the initiation factor responsible for guanine nucleotide exchange on eIF-2. Cell-free translation systems from unfertilized eggs are stimulated by added eIF-2B, although this dependency is rapidly lost in translation systems prepared at various times following fertilization. Cell-free translation systems prepared from unfertilized eggs show significantly lower eIF-2B activities than those prepared from 2-h embryos. However, the provision of an NADPH regeneration system significantly stimulates eIF-2B activity in egg extracts and, in addition, stimulates both binding of initiator tRNA to the small ribosomal subunit and protein synthetic activity. These data suggest that the activation of eIF-2B following fertilization reflects the fertilization-induced increase in NADPH levels.     

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.     

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.     

Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin

Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. β-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0–40% ammonium sulfate cut of intiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40–50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.     

Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle.

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.     

Comparison of cytoplasmic informosome proteins with RNA-binding proteins and translation initiation factors from rabbit reticulocytes.

Using one-and two-dimensional electrophoresis, the free and polyribosomal informosome proteins and a preparation of total RNA-binding proteins from rabbit reticulocytes were compared. It was shown that the major proteins of free and polyribosomal informosomes are similar only to the minor components of RNA-binding proteins. On the other hand, the major RNA-binding proteins, two of which are elongation translation factors EF-1L and EF2, can be present in informosome preparations only as minor components. The major proteins of polyribosomal informosomes do not coincide in terms of electrophoretic mobility with initiation factors eIF-2, eIF-2A, eIF-3, eIF-4A and eIF-4B. The major proteins of free informosomes differ in their electrophoretic mobility from initiation factors eIF-2A, eIF-4A and eIF-4B as well as from the alpha- and beta-subunits of initiation factor eIF-2.     

Identification and quantitation of levels of protein synthesis initiation factors in crude HeLa cell lysates by two-dimensional polyacrylamide gel electrophoresis

Protein synthesis initiation factors in purified preparations and in crude lysates of HeLa cells were fractionated by two-dimensional polyacrylamide gel electrophoresis in order to characterize their molecular forms. Specific spots in the complex cytoplasmic protein gel pattern which corresponded to the initiation factor proteins were identified by co-migration of purified initiation factors with 35S-labeled cell lysates, partial proteolytic digestion mapping, and immunoblotting analysis using antisera or affinity-purified antibodies to the initiation factors. Spots identified as eukaryotic initiation factor (eIF) 2 alpha, eIF-2 beta, eIF-2 gamma, eIF-4A, and four eIF-3 proteins of less than 50,000 Da corresponded to moderately abundant lysate proteins. Minor isoelectric variant forms of eIF-2 beta, eIF-2 gamma, and eIF-4A were detected by immunoblot analysis of lysate proteins, suggesting either covalent modification of these factor proteins or contaminating antibodies. eIF-2 beta and eIF-4B were present in at least two isoelectric forms, confirming covalent modification of these proteins. The cellular levels of the initiation factor proteins were measured by excising and counting radioactivity in gel-resolved spots corresponding to factors in lysates labeled in vivo. The individual factor protein abundancies span nearly a 10-fold range, from 1.1 to 9.8 million molecules/cell. The factor to ribosome ratio for eIF-2 was 0.8, for the average eIF-3 protein about 0.6, and for eIF-4A it was significantly higher at 3.0.     

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.     

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.     

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.     

Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors

Three mammalian eukaryotic initiation factors (eIF) are required for the ATP-dependent binding of mRNA to the 40 S ribosomal subunit. These three factors, eIF-4A, eIF-4B, and eIF-4F, have also been isolated from wheat germ. Three assays were used to measure the ability of the wheat germ factors to interact with and/or substitute for the mammalian factors. Two assay systems were used to measure partial reactions involving the interaction of the three factors, ATP, and mRNA: 1) RNA-dependent ATP hydrolysis and 2) cross-linking of the factors to the 5' cap of oxidized mRNA. A third assay system was used to measure the ability of the factors to support initiation of protein synthesis. The results of the ATP hydrolysis and cross-linking experiments indicate that the wheat germ factors can interact with or substitute for the mammalian factors. Wheat germ eIF-4A appears to be functionally equivalent to mammalian eIF-4A. Wheat germ eIF-4B and eIF-4F appear to be isozymes possessing functions similar to mammalian eIF-4F. Wheat germ eIF-4B does not appear to be a functional equivalent to the mammalian eIF-4B. In a complete translation system from wheat germ, mammalian factors partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system.     

Comparative analysis of phosphorylation of translational initiation and elongation factors by seven protein kinases

Four initiation factors (eIF-2, -3, -4B, and -4F), previously shown to be phosphorylated in vivo, are each phosphorylated to a significant extent in vitro (greater than 0.3 mol of phosphate/mol of factor) by at least three different protein kinases. An S6 kinase from liver, an active form of protease-activated kinase II which modifies the same sites on S6 as those phosphorylated in vivo in response to mitogens, phosphorylates the beta subunit of eIF-2, eIF-3 (p120-p130), eIF-4B, and eIF-4F (p220). The Ca2+, phospholipid-dependent protein kinase phosphorylates eIF-2 beta, eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220, p25). The cAMP-dependent protein kinase significantly modifies eIF-4B and, to a lesser extent, eIF-3 (p130). Casein kinase I incorporates phosphate only into eIF-4B, but to a limited extent. Casein kinase II phosphorylates eIF-2 beta, eIF-3 (p170, p120), and eIF-4B, while protease-activated kinase I modifies eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220). The mitogen-stimulated S6 kinase from 3T3-L1 cells, activated in response to insulin, does not phosphorylate any of the initiation factors. There is no significant incorporation of phosphate into eIF-2 alpha or -gamma, eIF-4A, eIF-4C, eIF-4D, EF-1, or EF-2 by any of the protein kinases examined. Phosphopeptide mapping of tryptic digests of the phosphorylated subunits shows that the individual protein kinases modify different sites. The sites phosphorylated in vitro reflect those modified in vivo as shown with eIF-4F in concomitant studies with reticulocytes treated with tumor-promoting phorbol ester (Morley, S.J., and Traugh, J. A. J. Biol. Chem., in press). Thus, we have identified multipotential protein kinases which modify four initiation factors phosphorylated in vivo and have shown that phosphorylation of these translational components can be coordinately regulated.     

Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition.

The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome-mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5' to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that involve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor-mediated stimulation of complex formation by a combination of eIF-4A, -4B, and -4F was observed, and was one which required the presence of ATP. This factor-mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5'-p-fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested.