Polypeptide Chain Initiation Research Articles

A Mechanism by which Adenovirus Virus-Associated RNAI Controls Translation in a Transient Expression Assay

The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.

A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.

The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF.

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.

Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2.

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.

The association of NADPH with the guanine nucleotide exchange factor from rabbit reticulocytes: a role of pyridine dinucleotides in eukaryotic polypeptide chain initiation.

The guanine nucleotide exchange factor (GEF) was purified to apparent homogeneity from postribosomal supernatants of rabbit reticulocytes by chromatography on DEAE-cellulose and phosphocellulose, fractionation by glycerol gradients, and chromatography on Mono S and Mono Q (Pharmacia). At the Mono S step GEF is isolated as a complex with the eukaryotic polypeptide chain initiation factor 2 (eIF-2) and is separated from this factor by column chromatography on Mono Q. An emission spectrum characteristic of a reduced pyridine dinucleotide was observed when GEF was subjected to fluorescence analysis. By both coupled enzymatic analysis and chromatography on reverse-phase or Mono Q columns, the bound dinucleotide associated with GEF was determined to be NADPH. The GEF-catalyzed exchange of eIF-2-bound GDP for GTP was markedly inhibited by NAD+ and NADP+. This inhibition was not observed in the presence of equimolar concentrations of NADPH. Similarly, the stimulation of ternary complex (eIF-2 X GTP X Met-tRNAf) formation by GEF in the presence of 1 mM Mg2+ was abolished in the presence of oxidized pyridine dinucleotide. These results demonstrate that pyridine dinucleotides may be directly involved in the regulation of polypeptide chain initiation by acting as allosteric regulators of GEF activity.

Formation of a translational inhibitor by interaction of phospholipid with the eukaryotic initiation factor 2.

The polypeptide chain initiation factor 2 (eIF-2) binds phospholipid (PL) and becomes a potent inhibitor of translation in hemin-supplemented reticulocyte lysates. This binding is markedly reduced by prior treatment of eIF-2 with N-ethylmaleimide. Although PL is probably bound by all three eIF-2 subunits, our results suggest that the inhibitory molecule is produced by binding to the alpha subunit because, functionally, PL binding has the same effect on eIF-2 as alpha-subunit phosphorylation. This is suggested by the following findings. (i) Like translational inhibition due to heme deficiency, inhibition by small amounts of the eIF-2 X PL complex is prevented by small amounts of the GDP exchange factor (GEF). (ii) In the presence of Mg2+, the GEF-catalyzed formation of a ternary complex (eIF-2 X GTP X Met-tRNAi in which Met-tRNAi is the eukaryotic initiator methionyl tRNA) is inhibited by eIF-2 X PL just as well as by eIF-2 alpha-subunit phosphorylation. (iii) Also in the presence of Mg2+, GEF is unable to catalyze the exchange of free GDP with eIF-2 X PL-bound GDP, as it fails to catalyze the exchange of free GDP with GDP that is bound to alpha-subunit-phosphorylated eIF-2. These observations suggest that, like alpha-subunit-phosphorylated eIF-2, eIF-2 X PL traps GEF in a nondissociable eIF-2 X PL X GEF complex, whereby GEF is no longer able to catalyze ternary complex formation and initiation is inhibited.

Inhibition of polypeptide chain initiation in Daudi cells by interferons. Evidence that activity of initiation factor eIF-2 and availability of mRNA are unimpaired

The accompanying paper [McNurlan & Clemens (1986) Biochem. J. 237, 871-876] shows that the inhibition of proliferation of Daudi cells by human interferons is associated with impairment of the overall rate of protein synthesis. We have examined whether two of the mechanisms which are believed to control translation in interferon-treated virus-infected cells may be responsible for the inhibition of protein synthesis during the antiproliferative response in these uninfected cells. Although the rate of polypeptide chain initiation is lower in interferon-treated Daudi cells, as indicated by the disaggregation of polysomes, there is no significant inhibition of activity of initiation factor eIF-2 or of [40 S . Met-tRNAf] initiation complex formation in cell extracts. The phosphorylation state of the alpha subunit of eIF-2 remains unaltered. There is no major decrease in mRNA content as a proportion of total RNA up to 4 days of interferon treatment, as judged by poly(A) content, although the amount of total mRNA/10(6) cells eventually declines. The mRNA present in extracts from interferon-treated cells remains translatable when added to an mRNA-dependent reticulocyte lysate system. We conclude that neither the interferon-inducible eIF-2 protein kinase pathway nor the 2',5'-oligo(adenylate)-ribonuclease L pathway are responsible for the inhibition of polypeptide chain initiation. Rather, the data suggest impairment at the level of formation of [80 S ribosome X mRNA] initiation complexes.

Use of an antibody to characterize and determine the role of the major Met-tRNA f deacylase from rabbit reticulocyte ribosomes

Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(α) results, secondarily, in the enzymatic deacylation of Met-tRNA f on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNA f deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNA f deacylase II and has no effect on Met-tRNA f deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNA f deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte lysate incubated in the absence of hemin or presence of hemin plus 0.1 μg/ml poly(I·C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNA f and increase the level of Met-tRNA f bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNA f in the 48 S complexes that accumulate.

Identification of ribosome-bound eukaryotic initiation factor 2.GDP binary complex as an intermediate in polypeptide chain initiation reaction.

Studies on the formation and release of the eukaryotic initiation factor (eIF)-2.GDP binary complex formed during eIF-5-mediated assembly of an 80 S initiation complex have been carried out. Incubation of a 40 S initiation complex with eIF-5, in the presence or absence of 60 S ribosomal subunits at 25 degrees C, causes rapid and quantitative hydrolysis of ribosome-bound GTP to form an eIF-2.GDP binary complex and Pi. Analysis of both reaction products by Sephadex G-200 gel filtration reveals that while Pi is released from ribosomes, the eIF-2.GDP complex remains bound to the ribosomal initiation complex. The eIF-2.GDP binary complex can however be released from ribosome by subjecting the eIF-5-catalyzed reaction products to either longer periods of incubation at 37 degrees C or sucrose gradient centrifugation. Furthermore, addition of a high molar excess of isolated eIF-2.GDP binary complex to a 40 S initiation reaction mixture does not cause exchange of ribosome-bound eIF-2.GDP complex formed by eIF-5-catalyzed hydrolysis of GTP. These results indicate that eIF-2.GDP complex is directly formed on the surface of ribosomes following hydrolysis of GTP bound to a 40 S initiation complex, and that ribosome-bound eIF-2 X GDP complex is an intermediate in polypeptide chain initiation reaction.

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.

Structural studies on the eukaryotic chain initiation factor 2 from rabbit reticulocytes and brine shrimp Artemia embryos. Phosphorylation by the heme-controlled repressor and casein kinase II.

In contrast to reticulocyte polypeptide chain initiation factor 2 (eIF-2), the Artemia factor retains activity in the presence of Mg2+ or after phosphorylation of its alpha-subunit by rabbit reticulocyte heme-controlled repressor (Mehta, H. B., Woodley, C. L., and Wahba, A. J. (1983) J. Biol. Chem. 258, 3438-3441). Furthermore, we have so far been unable to demonstrate a requirement for a GDP/GTP nucleotide exchange factor with Artemia eIF-2. In order to explain these differences we compared the structure of eIF-2 from Artemia and rabbit reticulocytes by using one- and two-dimensional phosphopeptide and iodopeptide maps. Partial trypsin digestion of the alpha-subunit of Artemia eIF-2 after phosphorylation by the heme-controlled repressor generates a 4000 Mr phosphopeptide. Upon extensive trypsin digestion, the two-dimensional phosphopeptide maps of the alpha-subunits for the reticulocyte and Artemia factors are indistinguishable, whereas the iodopeptide maps are different. In addition, immunoblotting indicates that there is no consistent cross-reactivity of the reticulocyte subunits with antibodies prepared in rabbits against the Artemia eIF-2 subunits. A casein kinase II activity was isolated from Artemia embryos that phosphorylates the beta-subunit of reticulocyte eIF-2, but specifically phosphorylates the alpha-subunit of eIF-2 preparations from several non-mammalian sources, including Artemia, yeast, and wheat germ embryos. Since this kinase phosphorylates a site distinct from that recognized by the heme-controlled repressor, and this phosphorylation does not alter the ability of Artemia eIF-2 to undergo nucleotide exchange, caution must be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in non-mammalian cells.

Effect of exercise on protein turnover in muscles of lean and obese mice

The effect of work-induced hypertrophy on skeletal muscle protein metabolism was studied in lean mice and in mice rendered obese with goldthioglucose. After tenotomy of the gastrocnemius muscle, the adaptative growth of soleus muscle was less pronounced in obese than in lean mice. Protein turnover was studied in the isolated soleus muscle 4 days after the operation. Tyrosine incorporation in total proteins and tyrosine release in the incubation medium (indices of protein synthesis and degradation) were increased by 3- and 2-fold in overloaded (i.e. work-induced hypertrophied) muscles of lean and obese mice, respectively, compared to the control muscles isolated from the non-operated leg. The qualitative modifications in the pattern of proteins synthetized from 35S-methionine were identical in both groups of mice. This increase in protein turnover in overloaded muscles results from an increased rate of polypeptide chain initiation (3-fold in lean mice, 2.2-fold in obese mice) without any modification of peptide chain elongation rates. Work-induced hypertrophy was also able to reverse the defect in amino acid uptake which is present in soleus muscles of GTG-obese mice. These results suggest that the work-induced increase of skeletal muscle protein turnover is diminished in obese mice.

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits.

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).

Control of polypeptide chain initiation by sugar phosphates.

Control of polypeptide chain initiation by sugar phosphates.

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity.

Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.

The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli

The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic + strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium. The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter. If glucose was restored to the medium at any time after shiftdown, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased. These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells. The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies. Low initiation frequencies represent increased “dwell” of the ribosomes at the initiation site before the commencement of polypeptide chain initiation. We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site.

Purification and characterization of a guanosine diphosphatase activity from calf liver microsomal salt wash proteins.

A potent guanosine diphosphatase activity that hydrolyzes GDP to 5'-GMP + Pi has been isolated and purified from the salt wash proteins of calf liver microsomes. The purified enzyme, a monomeric protein of approximate Mr 46,000, possesses nucleotide substrate specificity since, among the nucleoside diphosphates and triphosphates tested, only GDP and UDP are hydrolyzed by the enzyme. The relative affinity of the enzyme for GDP is, however, much higher than for UDP. The effect of the enzyme on the binary complex formed between eukaryotic initiation factor 2 (eIF-2) and GDP has also been investigated. The enzyme neither hydrolyzes GDP bound to eIF-2 nor catalyzes the exchange of eIF-2-bound GDP with GTP even in the presence of Met-tRNAf. The enzyme, therefore, is presumably not involved in recycling of eIF-2 in eukaryotic polypeptide chain initiation reaction. The possible biological function of the enzyme in maintaining the cellular pool of GTP-GDP is discussed.

Studies on the activation of the heme-stabilized translational inhibitor of reticulocyte lysates by oxidized glutathione and NADPH depletion

The translational inhibition produced by addition of oxidized glutathione (GSSG) to hemin-containing reticulocyte lysates and the accompanying phosphorylation of the α subunit of the polypeptide chain initiation factor eIF-2 can be prevented or reversed by NADPH generators, including glucose 6-phosphate, deoxyglucose 6-phosphate, fructose 6-phosphate, NADPH itself, and also by dithiols, e.g., dithiothreitol, but not by reduced glutathione (GSH) or other monothiols, e.g., 2-mercaptoethanol. The same is true of the inhibition caused by addition of glutamate dehydrogenase, α-ketoglutarate, and NH 4 +, which may be entirely due to NADPH depletion via the reaction α-ketoglutarate + NH 3 + NADPH + H + glutamate + NADP + + H 2O. Although GSSG can also deplete NADPH via the reaction catalyzed by glutathione reductase (GSSG + NADPH + H + 2 GSH + NADP +) there are indications that another mechanism is operative. In sharp contrast to the inhibition caused by GSSG or by NADPH depletion is the translational inhibition produced by heme deficiency which is prevented by neither NADPH generators nor dithiols. This indicates that the proinhibitor form of the heme-controlled translational inhibitor may be activated by two entirely different, unrelated mechanisms, one of which appears to involve oxidation of the enzyme.

Translational control by adenovirus: lack of virus-associated RNAI during adenovirus infection results in phosphorylation of initiation factor eIF-2 and inhibition of protein synthesis.

The dl331 mutant of adenovirus serotype 5 fails to produce virus-associated (VA) RNAI, and cells infected with this mutant do not synthesize proteins efficiently at late times in infection. The translational defect occurs at the level of polypeptide chain initiation, and cell-free extracts prepared from dl331-infected cells exhibit the defect observed in vivo. Addition of either eukaryotic initiation factor 2 (eIF-2) or guanine nucleotide exchange factor (GEF) to these cell-free extracts restores translational activity, with GEF functioning more efficiently in this regard. These results suggest that cells infected with the dl331 mutant develop a translational block at the level of GEF-catalyzed guanine nucleotide exchange and that this block is most likely established through phosphorylation of the alpha subunit of eIF-2. In the present investigation we show that endogenous HeLa cell GEF activity is significantly reduced in cells infected with the dl331 mutant. Further, in contrast to cells infected with wild-type serotype 2 adenovirus, dl331-infected cells contain increased eIF-2 alpha kinase activity. These results indicate that VA RNAI plays a role in suppressing eIF-2 alpha kinase activity during adenovirus infection of HeLa cells.

Formation and release of eukaryotic initiation factor 2 X GDP complex during eukaryotic ribosomal polypeptide chain initiation complex formation.

The formation and release of an eukaryotic initiation factor (eIF)-2 X GDP binary complex during eIF-5-mediated assembly of an 80 S ribosomal polypeptide chain initiation complex have been studied by sucrose gradient centrifugation analysis. Isolated 40 S initiation complex reacts with eIF-5 and 60 S ribosomal subunits to form an 80 S ribosomal initiation complex with concomitant hydrolysis of an equimolar amount of bound GTP to GDP and Pi. Sucrose gradient analysis of reaction products revealed that GDP was released from ribosomes as an eIF-2 X GDP complex. Evidence is presented that eIF-5-mediated hydrolysis releases the GTP bound to the 40 S initiation complex as an intact eIF-2 X GDP complex rather than as free GDP and eIF-2 which subsequently recombine to form the binary complex. Furthermore, formation and release of eIF-2 X GDP from the ribosomal complex do not require concomitant formation of an 80 S initiation complex since both reactions occur efficiently when the 40 S initiation complex reacts with eIF-5 in the absence of 60 S ribosomal subunits. These results, along with the observation that the 40 S initiation complex formed with the nonhydrolyzable analogue of GTP, 5'-guanylylmethylene diphosphonate, can neither join a 60 S ribosomal subunit nor releases ribosome-bound eIF-2, suggest that following eIF-5-mediated hydrolysis of GTP bound to the 40 S initiation complex, both Pi and eIF-2 X GDP complex are released from ribosomes prior to the joining of 60 S ribosomal subunits to the 40 S initiation complex.