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1.
A. Majumdar R. Roy A. Das A. Dasgupta N.K. Gupta 《Biochemical and biophysical research communications》1977,78(1):161-169
The peptide chain initiation factor, EIF-2 has been partially purified from the 0.5 M KCl ribosomal wash. The molecular weight of EIF-2 is approximately 450,000. The purified EIF-2 preparation promotes the dissociation of the ternary complex, Met-tRNAf·EIF-1·GTP in the presence of Mg++ and is also required along with EIF-1 for AUG-directed Met-tRNAf binding to 40S ribosomes. 相似文献
2.
A protein synthesis initiation inhibitor, TDI has been partially purified from the reticulocyte cell-supernatant. TDI inhibits the dissociation of the ternary complex, Met-tRNAf·EIF-1·GTP and also Met-tRNAf binding to 40S ribosomes. TDI inhibition requires Mg++ and the inhibition is also observed when GTP is replaced by a non-hydrolyzable analog, GMP-PNP. 相似文献
3.
A Dasgupta R Roy S Palmieri A Das R Ralston N K Gupta 《Biochemical and biophysical research communications》1978,82(3):1019-1027
The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNAf·EIF-1·GTP in the absence of Mg++ and the preformed complex is stable to Mg++. However, with homogeneous preparations of EIF-1, addition of Mg++ during the initial formation of the ternary complex strongly inhibits the complex formation.A heat stable dialyzable factor (EIF-11) which mostly remains associated with the high molecular weight protein complex, EIF-2 (TDF) during purification of the peptide chain initiation factors, has been purified using a phenol extraction procedure. EIF-11 restores the Met-tRNAf binding activity of EIF-1 in the presence of 1 mM Mg++; in the presence of EIF-11, Met-tRNAf binding by EIF-1 shows a sharp Mg++ optimum around 1 mM. EIF-11 is heat stable, alkali stable, dialyzable and pronase sensitive. The same EIF-11 preparation also strongly inhibits Met-tRNAf binding to EIF-1 in the absence of Mg++ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system. 相似文献
4.
James S. Hutchison Kivie Moldave 《Biochemical and biophysical research communications》1981,99(2):722-728
The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNAf into eIF-2·Met-tRNAf·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNAf required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature. 相似文献
5.
Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors 总被引:39,自引:0,他引:39
The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [125I]mRNA, [3H]Met-tRNAf, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNAf to the 40 S ribosomal subunit. The initial Met-tRNAf binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented. 相似文献
6.
The crude soluble fraction of rat liver cytoplasm promotes the binding of acetylphenylalanyl-tRNA but not of Met-tRNAf to 40S subunits derived from 80S ribosomes. A protein has been extensively purified from the soluble fraction that catalyzes the template-dependent, GTP-independent binding of Met-tRNAf, acetylphenylalanyl-tRNA and phenylalanyl-tRNA but not Met-tRNAm. Purification involves fractionation with ammonium sulfate and chromatography on calcium phosphate gel, DEAE-Sephadex, carboxymethyl cellulose and Sephadex G-200. The optimum Mg2+ concentration for the binding reaction with Met-tRNAf is between 6 and 8 mm and the optimum temperature is between 10 and 15 °C. The complex formed as a result of the interaction between 40S subunits, acetylphenylalanyl-tRNA and poly(U) is functional; acetylpolyphenylalanine is synthesized when the isolated 40S-poly(U)·acetylphenylalanyl-tRNA complex is incubated with 60S subunits, phenylalanyl-tRNA, elongation factors and GTP.The crude cytoplasmic fraction, which does not stimulate the binding of Met-tRNAf, inhibits the purified factor-promoted binding of this substrate; the factor-independent, high magnesium ion-stimulated binding of Met-tRNAf to 40S subunits is also inhibited. The inhibitory activity can be resolved from the binding factor and is extensively purified by chromatography on calcium phosphate gel and carboxymethyl Sephadex and by electrofocusing. In the presence of 40S subunits, crude and purified preparations of the inhibitory activity hydrolyze Met-tRNAf but not Met-tRNAm or acetylphenylalanyl-tRNA. Free Met-tRNAf is not hydrolyzed. Incubation of hydrolase-containing preparations with the preformed 40S-·Met-tRNAf complex results in the rapid and extensive breakdown of the complex with release of acid-insoluble methionine; the formation of an 80S·substrate complex, by the addition of 60S subunits, protects particle-bound Met-tRNAf. 相似文献
7.
The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNAfMet. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNAfMet (retic., ). Other aminoacyl tRNAs tested including fMet-tRNAfMet (retic., ), Phe-tRNA (), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNAfMet forms the initiation complex Met-tRNAfMet:IF1:GTP (2), and in this ternary complex Met-tRNAfMet is not degraded by the deacylase. Met-tRNAfMet binds to IF1 independent of GTP, and in this complex, this Met-tRNAfMet is degraded by the deacylase.Prior incubation of f1 with Met-tRNAfMet (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNAfMet (retic.) was pre-incubated with peptide chain initiation factors. 相似文献
8.
Evan A. Stringer Asok Chaudhuri Umadas Maitra 《Biochemical and biophysical research communications》1977,76(2):586-592
A highly purified preparation of the eucaryotic initiation factor eIF-2 from calf liver which forms a ternary complex with GTP and Met-tRNAfMet also exhibits a potent GDP binding activity. The factor preparation specifically forms a binary complex with GDP, other ribonucleoside diphosphates and GTP are inactive. Evidence is presented indicating that the GTP-dependent Met-tRNAfMet binding and binary complex formation with GDP are mediated by the same protein which has an apparent molecular weight of 67,000 as judged by glycerol density gradient centrifugation. 相似文献
9.
The initiation factor eIF-2 that specifically binds Met-tRNAf and GTP in ternary complex (eIF-2. GTP. Met-tRNAf) has been purified to apparent homogeneity from wheat germ ribosomal salt wash. The purified factor exhibits a sedimentation coefficient of 5 · 5S and an aggregate molecular weight of 122000-daltons for the native protein.A preliminary account of this work was presented at the 66th Annual (1982) Meeting of the Federation of American Societies for Experimental Biology; Fed Proc 41, 1040. 相似文献
10.
Wolf-Jürgen Buhl Thomas F. Sarre Kurt Hilse 《Biochemical and biophysical research communications》1980,93(3):979-987
From a rabbit reticulocyte postpolysomal supernatant a fraction has been isolated which is enriched in ribosomal particles sedimenting at 50S. This fraction is efficiently in vitro translated predominantly into α-globin. Besides the RNAs and proteins of the small ribosomal subunit the 50S particle contains α-globin mRNA and additional high molecular weight proteins, most of which correspond to polypeptides of the initiation factors eIF-2 and eIF-3. The 50S particle may represent a native [mRNA·40S·eIF′s·Met-tRNAf·GTP] complex which may occur in vivo as a translatable intermediate in the initiation sequence. 相似文献
11.
Mutually exclusive binding of messenger RNA and initiator methionyl transfer RNA to eukaryotic initiation factor 2 总被引:3,自引:0,他引:3
Eukaryotic initiation factor 2 (eIF-2), purified to at least 98% homogeneity as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and containing no detectable amounts of eukaryotic initiation factor 4B (eIF-4B), is active both in the binding of Met-tRNA and in the binding of globin mRNA. The mRNA-binding activity is completely sensitive to competitive inhibition by Met-tRNA, provided GTP is present, but not by uncharged tRNA. By contrast, binding of mRNA to partially purified eIF-4B is not inhibited by Met-tRNAf. These results establish that the only mRNA-binding component in the eIF-2 preparation is eIF-2 itself, and show that a given molecule of eIF-2 can either bind to a molecule of mRNA, or form a ternary complex with Met-tRNAf and GTP, but cannot do both at once. 相似文献
12.
Bernhard Erni Theophil Staehelin 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,740(4):373-378
Binding of the Met-tRNAMetf·eIF-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNAMetf exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNAMetf binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNAMetf mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur. 相似文献
13.
Ternary complex formation was studied in reticulocyte lysate supernatants and using rat liver eukaryotic initiation factor-2 (eIF-2) preparations. Haem-deficiency reduced the rate of formation of ternary (Met-tRNAf · GTP · eIF-2) complexes by the eIF-2 in reticulocyte supernatants, the reduction being more marked when complex formation was assayed in the absence of GTP-regenerating capacity. Pretreatment with the haem-controlled repressor (HCR) reduced the rate of ternary complex formation by crude (liver) eIF-2. In contrast, complex formation by an almost homogeneous eIF-2 preparation was unaffected by HCR: sensitivity to HCR was however restored by a factor which catalyses exchange of guanine nucleotides bound to eIF-2. 相似文献
14.
S Mizuno 《Archives of biochemistry and biophysics》1977,179(1):289-301
When a reticulocyte lysate, supplemented with hemin, was warmed at 42 °C, its protein-synthesizing activity was greatly decreased. This was accompanied by the reduced formation of the 40 S·Met-tRNAf initiation complex. This complex preformed at 34 °C, however, was stable and combined with added globin mRNA and the 60 S ribosomal subunit to form the 80 S complex at the elevated temperature. When the ribosome-free supernatant fraction of lysates was warmed at 42 °C with hemin and then added to the fresh lysate system, it inhibited protein synthesis by decreasing the formation of the 40 S complex. This decrease in protein synthesis by warmed lysates or warmed supernatant could be overcome by high concentrations of GTP and cyclic AMP. This effect of GTP and cyclic AMP was antagonized by ATP. The results indicate that the inactivation of protein synthesis by the lysate warmed at 42 °C is due to the formation of an inhibitor in the supernatant. The ribosomal KCl extract prepared from the lysate that had been warmed at 34 °C and then incubated at this temperature for protein synthesis supported protein synthesis by the KCl-washed ribosome at both 34 and 42 °C. On the contrary, the extract from lysates that had been warmed at 42 °C and then incubated at 34 °C could not support protein synthesis at 42 °C, although it was almost equally as promotive as the control extract in supporting protein synthesis at 34 °C. The results indicate that the factor which can protect protein synthesis against inactivation at 42 °C is itself inactivated in lysates warmed at 42 °C. However, the activity of this extract to support formation of the ternary complex with Met-tRNAf and GTP was not reduced. Native 40 S ribosomal subunits isolated from lysates that had been warmed at 42 °C and then incubated for protein synthesis indicated that the quantity of subunits of density 1.40 g/cm3 in a CsCl density gradient were decreased while those of density 1.49 g/cm3 were increased. The factor-promoted binding of Met-tRNAf to the 40 S subunit of lower density from the warmed and unwarmed lysates was equal, suggesting that the ribosomal subunit was not inactivated. These results were discussed in terms of the action of the inhibitor formed in the supernatant at 42 °C, which may inactivate a ribosomal factor essential for protein synthesis initiation. 相似文献
15.
P. Ghosh-Dastidar B.B. Goswami A. Das P. Das N.K. Gupta 《Biochemical and biophysical research communications》1981,99(3):946-953
Vaccinia viral core inhibits protein synthesis in reticulocyte lysates. In partial reactions using micrococcal nuclease treated reticulocyte lysates, the viral core inhibits Met-tRNAf binding to 40S ribosomes in response to physiological mRNAs such as globin mRNA, cowpea mosaic viral RNA, and brome mosaic viral RNA but not in response to a trinucleotide codon, AUG. The core has also no effect on Met-tRNAf binding to 40S ribosomes in a partial reaction using partially purified peptide chain initiation factors and AUG codon.The present observation of preferential inhibition by vaccinia viral core of Met-tRNAf·40S initiation complex formation with physiological mRNAs and not with an artificial mRNA such as AUG codon, suggests that the viral core inhibits some step(s) in peptide chain initiation involved in the recognition of structural feature(s) unique to physiological mRNAs. 相似文献
16.
Initiation of protein synthesis: evidence for messenger RNA-independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit 总被引:45,自引:0,他引:45
This paper shows that reticuloeyte lysates contain 40 S/Met-tRNAf complexes which are intermediates in the initiation of protein synthesis before the involvement of messenger RNA. More than one third of the native 40 S subunits in the lysate exist as these complexes during periods of linear protein synthesis, but less than a tenth are associated with mRNA.The 40 S/Met-tRNAf complexes disappear in some situations in which initiation is inhibited (by double-stranded RNA, oxidized glutathione, or in the absence of added haemin), but persist in the presence of other inhibitors (e.g. aurintricarboxylate or poly(I)). Inhibitors of chain elongation had little effect on the amount of these complexes.The Met-tRNAf in the 40 S complexes appears to exchange readily with free Met-tRNAf; when lysates were preincubated with sparsomycin or diphtheria toxin and then incubated with [35S]Met-tRNAf, the native 40 S subunits were the only ribosomal particles labelled. This experimental system was used to examine whether 40 S/Met-tRNAf complexes could interact with mRNA; various mRNAs were added shortly after or at the same time as the [35S]Met-tRNAf. This resulted in a conversion of the 40 S/Met-tRNAf complexes into 80 S complexes, which appeared to be true initiation complexes since they were capable of translating the first two codons of the added mRNA. The mRNA-dependent formation of these 80 S complexes was completely inhibited by 0.1 mM-aurintricarboxylate, but the association of Met-tRNAf with the 40 S subunits was not prevented.The 40 S/Met-tRNAf complexes also participated in initiation on endogenous mRNA, and it was shown that the Met-tRNAf in this complex was used in preference to free Met-tRNAf in this process.We propose that the first step in the initiation of protein synthesis in the reticuloeyte lysate is the formation of a 40 S/Met-tRNAf complex. In the second stage the complex binds mRNA at the correct initiation site and, after joining with a 60 S subunit, an 80 S/Met-tRNAf/mRNA initiation complex is formed. 相似文献
17.
M J Clemens C O Echetebu V J Tilleray V M Pain 《Biochemical and biophysical research communications》1980,92(1):60-67
Phosphocellulose chromatography of initiation factor eIF-2 from rat liver separates it from a protein fraction which is highly stimulatory for [eIF-2.GTP.Met-tRNAf] ternary complex formation. Evidence is presented which indicates that this stimulatory fraction contains a specific GDPase activity. eIF-2 dependent formation of 40S ribosomal initiation complexes is also enhanced by the GDPase preparation. The enzyme may play a role in the recycling of eIF-2 by removing inhibitory GDP which is generated during 80S initiation complex formation. 相似文献
18.
Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors 总被引:33,自引:0,他引:33
We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNAf · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP. 相似文献
19.
Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg2+ concentrations examined, binding of the putative initiator Met-tRNAi to 40 S subunits is greater than that observed with Met-tRNAm. Also, binding of Met-tRNAi to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNAi complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNAi is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNAMet) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNAi binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNAi and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNAi is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes. 相似文献
20.
Treatment of formylmethionine tRNA with sodium bisulfite produces six C → U base changes in the tRNA structure. Four of these modifications have no effect on the ability of tRNAfMet to be aminoacylated or formylated. Prior to bisulfite treatment, Met-tRNAfMet is not able to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. After bisulfite treatment, a large portion of the modified tRNA is bound as T-GTP-Met-tRNAfMet. Formylation of bisulfite-modified Met-tRNAfMet completely eliminates T factor binding. Unmodified tRNAfMet is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5′ terminus. Bisulfite-catalyzed conversion of this unpaired C1 to U1 results in formation of a normal U1-A73 base pair at the end of the acceptor stem. It is likely that this structural alteration is responsible for the recognition of bisulfite-modified Met-tRNAfMet by T factor. 相似文献