Eukaryotic Protein Synthesis Initiation Research Articles

Initiation of mammalian protein synthesis: Dynamic properties of the assembly process in vitro

Binding of the Met-tRNA Met f·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-tRNA Met f exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNA Met f binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNA Met f mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.

Protein synthesis eukaryotic initiation factors 4A and 4B are not altered by poliovirus infection of HeLa cells.

Infection of HeLa cells by poliovirus results in the inhibition of translation of capped cellular mRNA. A plausible mechanism for this inhibition is that the structure of one or more initiation factors involved in the recognition of capped mRNA is altered. Eukaryotic initiation factor (eIF) 4A and eIF-4B are implicated in mRNA binding to 40 S ribosomal subunits and can be cross-linked to oxidized capped mRNA. We examined these factors in HeLa cell lysates by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. No alterations in the number of molecules/cell, in the molecular size, or in extents of covalent modification were detected when lysates from infected and mock-infected cells were compared. The integrity of eIF-2 and several eIF-3 polypeptides was also examined and likewise no alterations were detected. The failure of the translational machinery to recognize capped mRNA therefore is not due to a change in the structure of these initiation factors.

The Control of the Rate of Initiation of Eukaryotic Protein Synthesis

Publisher Summary This chapter discusses the control of the rate of initiation of eukaryotic protein synthesis. Control of the rate of initiation of protein synthesis is manifested at many different occasions during the lifetime of an average eukaryotic cell. During the cell cycle, there is no initiation of protein synthesis in mitosis. In the late S phase and in G2 phase, a gradual diminishing of initiation occurs. Under physiological stress—such as serum deprivation, amino-acid starvation, and hyperthermic treatment—the initiation rate is lowered substantially. The model for the control of the rate of initiation implies that when a complex is formed between eIF-2 and eRF, a high rate of initiation is achieved, whereas conditions that impair complex formation inevitably cause a low initiation rate. Reduced eIF-2 displays a much higher activity in lysate systems than oxidized eIF-2.

Base-pair formation between 18 S ribosomal RNA and globin mRNA during initiation of protein synthesis in vitro

A stable complex between 18 S rRNA and globin mRNA has been isolated from 40 S initiation complexes in the reconstituted reticulocyte cell free system. This complex is only formed under the conditions which also lead to an initiation complex active in protein synthesis. The mRNA—18 S rRNA interactions has properties compatible with base-pairing. This observation is discussed in the context with other, in part controversial, observations relating to base pairing as a step in initiation of eukaryotic protein synthesis.

Roles of Eukaryotic Initiation Factor 2 Ancillary Factors in the Regulation of Eukaryotic Protein Synthesis Initiation

This chapter discusses a possible mechanism of protein synthesis inhibition by eukaryotic initiation factor-2 (eIF-2) kinases, heme-regulated protein synthesis inhibitor, and double-stranded RNA-activated inhibitor; it discusses the roles of four eIF-2 ancillary protein factors in the complex regulation of protein synthesis initiation in animal cells. The loss of interaction of eIF-2α(P) with Co-eIF-2B and Co-eIF-2C has been suggested as the mechanism of protein synthesis inhibition by eIF-2 kinases. However, a direct evidence that the formation of eIF-2α(P) and consequent loss of interaction of eIF-2α(P) with Co-eIF-2B and Co-eIF-2C are the sole cause of protein synthesis inhibition in heme-deficient reticulocyte lysates is still lacking. The four eIF-2 ancillary factors, Co-eIF-2A, Co-eIF-2B, Co-eIF-2C, and reversal factor, have been characterized by their effects on eIF-2 activity in vitro . The requirement for Co-eIF-2A for protein synthesis in reticulocyte lysates is shown by using antibodies. It has not yet been possible to reconstitute from purified components an efficient protein-synthesizing system with which to examine the requirements for specific factors in overall peptide chain initiation.

Initiation of eukaryotic protein synthesis

The initiation of protein synthesis is defined as the sequence of events, which leads to an 80 S . MettRNA . mRNA complex. Several non-ribosomal proteins are required for the formation of this 80 S initiation complex and they are called eIF-1, -2, etc. (eukaryotic initiation factor). They are listed in table 1, with the effect they have on protein synthesis and on partial reactions thereof, as desiribed below. An initiation factor is defined as a protein which stimulates one or more of these, and only these reactions, and which is released after the completion of an 80 S initiation complex, in contrast with a ribosomal protein, which remains an integral part of the ribosome during all stages of protein synthesis. Dissociation of 80 S ribosomes is a prerequisite for the initiation of eukaryotic protein synthesis, since the initial binding of Met-tRNA occurs on a 40 S subunit and not on an 80 S ribosome. Spontaneous

The purification and characterization of multiple forms of protein synthesis eukaryotic initiation factors 2, 3, and 5 from rabbit reticulocytes.

Eukaryotic initiation factors 2, 3, and 5 (eIF-2, eIF-3, and eIF-5) each were purified or fractionated into a number of forms which differ in protein mass or composition. The various factor polypeptides were analyzed by partial protease digestion and the fragmentation patterns were compared to identify proteins related in primary structure. The digestion method was made more sensitive by prior radiochemical labeling of the proteins by iodination with 125I. At least four molecular weight forms of eIF-5 were found which ranged from 168,000 to 128,000. The lower molecular weight forms, nevertheless, were biologically active in in vitro assays. Analysis of a variety of eIF-3 preparations indicates that the presence or abundance of especially the higher molecular weight subunits varies considerably. Many of these components are related to one another in primary structure; five of the subunits greater than Mr = 90,000 appear to be derived from the 210 kilodalton subunit by limited proteolysis. Analysis of eIF-2 polypeptides shows that the alpha, beta, and gamma subunits give distinctively different partial protease digestion patterns and that some preparations contain a minor component (gamma') related to the gamma subunit which could be misidentified as the beta subunit. The results indicate that initiation factors may be cleaved into different active forms by proteases acting either in the intact cell or during the factor isolation and purification. Addition of the protease inhibitor, phenylmethanesulfonyl fluoride, prevents some, but not all of the proteolysis.

Identification of neighbouring components in the quaternary eukaryotic protein synthesis initiation complex, eIF-2.GTP.Met-tRNAf.small ribosomal subunit.

Identification of neighbouring components in the quaternary eukaryotic protein synthesis initiation complex, eIF-2.GTP.Met-tRNAf.small ribosomal subunit.

Mode of action of protein synthesis initiation factor eIF-1 from rabbit reticulocytes

Initiation of eukaryotic protein synthesis is mediated by a set of protein factors, purified using predominantly rabbit reticulocytes and Krebs II ascites cells. At least 8 initiation factors with Mr 15000-700000 could be described. This elaborate work was followed by the functional characterization of the individual factors which provided considerable insight in the specific contribution of each factor during the assembly of an initiation complex, reviewed.

Synthesis and translation of mRNA containing 5'-terminal 7-ethylguanosine cap.

Reovirus mRNA synthesis in vitro by the virion-associated RNA polymerase was only slightly (10 to 15%) diminished in the presence of 2 mM S-adenosylethionine. However, methyl group transfer from S-adenosylmethionine (0.05 mM) to the 5'-terminal cap structure, m7GpppGm in this mRNA was markedly inhibited (80%) under these conditions. Replacement of S-adenosylmethionine by S-adenosylethionine (5 mM) yielded mRNAs containing mainly (70%) 5'-terminal e7GpppGe and e7GpppG, but some of the products were unalkylated (5'-GpppG, ppG). The ethylated mRNAs, but not the unalkylated molecules, bound to wheat germ ribosomes and were translated essentially as well as the corresponding methylated mRNAs in wheat germ extracts and in nuclease-treated rabbit reticulocyte lysates. Protein synthesis directed by ethylated mRNAs in wheat germ extract was 80% decreased by 0.1 mM m7GMP. Under conditions of limited initiation, methylated mRNA bound to wheat germ ribosomes preferentially as compared to ethylated mRNA. The results document for the first time the synthesis of ethylated mRNA and support the hypothesis that N7-alkylation of the 5'-guanosine in caps, rather than methylation itself, is important for the enhancing effect of cap on the initiation of eukaryotic protein synthesis.

Phosphorylation of eukaryotic protein synthesis initiation factors.

Phosphorylation of eukaryotic initiation factors was examined both in intact cells and in vitro with purified components. Intact rabbit reticulocytes were incubated in a medium containing[32P]phosphate, and eight initiation factors were isolated and partially purified. The purified factors were analyzed on dodecyl sulfate/polyacrylamide gels and compared with highly purified nonradioactive factors. Significant amounts of radioactivity were found associated with initiation factors eIF-2, polypeptide 2 (molecular weight 53,000); eIF-3, polypeptides 2 and 4 (molecular weights 110,000 and 67,000); and eIF-4B. Purfied initiation factors from rabbit reticulocytes were also treated in vitro with [gamma-32P]ATP and a cyclic AMP-independent protein kinase isolated from rabbit erythrocytes. Only the factor polypeptides phosphorylated intracellularly were phosphorylated in vitro. The results suggest that the cyclic AMP-independent protein kinase is responsible for the phosphorylation of specific initiation factors in cells active in protein synthesis and that it may play a role in regulating translation.

Inhibition of initiation, elongation, and termination of eukaryotic protein synthesis by trichothecene fungal toxins.

The 12,13-epoxytrichothecenes, specific inhibitors of protein synthesis in eukaryotes, can be subdivided further in terms of their mode of action. In addition to the I-type (initiation inhibitors) and E-types (elongation inhibitors), we found that some E-types apparently exhibit inhibition of chain termination at low concentrations. The nature of substituents on C4 may determine the type of inhibitory activity observed.

Functional identity of a mouse ascites and a rabbit reticulocyte initiation factor required for natural mRNA translation.

INITIATION of eukaryotic protein synthesis involves the assembly of a functional initiation complex from components including ribosomal subunits, initiator tRNA (Met-tRNAF), initiation factors, messenger RNA, ATP and GTP1–3. Much research in this area has centred on the possible control of eukaryotic translation at the level of polypeptide initiation, as this step seems to be rate-limiting in the overall synthetic reaction4,5. Some studies have suggested the existence of tissue-specific initiation factors6, others have described initiation factors which apparently discriminate between eukaryotic mRNAs7. Work in this laboratory led to the purification of an initiation factor of molecular weight 53,000 from the supernatant fraction of Krebs II ascites cells8. This factor (IFEMC) was shown to be required for the translation of encephalomyocarditis virus RNA (EMC RNA) but not for globin mRNA translation in a fractionated ascites cell-free system9. Other studies on the translation of mRNAs injected into living Xenopus laevis oocytes10, and on the translation of various mRNAs in several crude eukaryotic protein-synthesising systems11 have suggested, however, that eukaryotic translation proceeds without controls exerted by specific initiation factors. We have adopted a comparative approach to this problem, examining the translation of various eukaryotic mRNAs in a fractionated cell-free system from Krebs II ascites cells, and testing the specificity of two mammalian factors reported to be required for the initiation of natural mRNA translation.

Nucleotide regulation of a eukaryotic protein synthesis initiation complex

Formation of a ternary initiation complex containing Met-tRNA f, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 · 10 −6 M. GDP is a potent competitive inhibitor of ternary complex formation with K i = 3.4 · 10 −7 M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with K GDP D = 3.0 · 10 −8 M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [ 3H] GDP or [ 3H] GTP from the nucleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.

Comparative studies on the properties of the eukaryotic protein synthesis initiation factor IF-I from several sources

Eukaryotic protein synthesis initiation factor IF-I has been purified from a variety of cells and tissues, and some of the properties of these preparations are compared. IF-I activity from all sources elutes close to the excluded volume of Sephadex G-200 columns, indicating similar, if not identical, molecular weights (350 000). There is little or no species or tissue specificity in the binding of the initiator tRNA from one source to IF-I purified from another source. The ability of all IF-I preparations to form a ternary complex with GTP and the initiator tRNA is inactivated by N- ethylmaleimide ; this sulfhydryl reagent also completely inactivates the rabbit reticulocyte IF-I-mediated binding of the initiator tRNA (rabbit liver) to rabbit reticulocyte 40-S ribosomal subunits. Thus, it appears that IF-I is an obligatory and ubiquitous protein synthesis initiation factor in the cytoplasm of all eukaryotes.

Paper electrophoresis and chromatography of oligopeptides with N-terminal methionine

Several electrophoretic and chromatographic systems are described for the separation of di- and tripeptides with N-terminal methionine. The most effective methods of separation are electrophoresis in barbital-triethylamine-acetic acid, pH 8.0 and chromatography in n-butanol-acetic acid-water, 18:2:5. Increased resolution may be obtained by using two or more systems in combination. These methods were intended to separate and identify the oligopeptides formed during the initiation of eukaryotic protein synthesis, but may also be applied to the separation of small peptides generally.

Initiation of Eukaryotic Protein Synthesis: Native 40S Subunits Bind Initiator Transfer Ribonucleic Acid before Associating with Messenger Ribonucleic Acid

Initiation of Eukaryotic Protein Synthesis: Native 40S Subunits Bind Initiator Transfer Ribonucleic Acid before Associating with Messenger Ribonucleic Acid

Initiation of Eukaryotic Protein Synthesis: [Met-tRNAf.40S Ribosome] Initiation Complex Catalysed by Purified Initiation Factors in the Absence of mRNA

Two mammalian initiation factors are needed to bind initiator Met-tRNAf to the 40S ribosome subunit in the absence of mRNA. One, purified from three different sources, catalyses specifically the mRNA-independent binding reaction.

Initiation of Eukaryotic Protein Synthesis

Initiation of Eukaryotic Protein Synthesis

The ternary complex of initiation factor IF-I, Met-tRNA Metf and GTP An aurintricarboxylate-sensitive intermediate in the initiation of eukaryotic protein synthesis

We have previously described an initiation factor, extensively purified from 0.5 M KCl extracts of rabbit reticulocyte polysomes, which forms a ternary 1:1 complex with the initiator tRNA [Met-tRNA Met f (rabbit liver)] and GTP. We now refer to this initiation factor as IF-I. When the ternary complex was separated from GTP and [ 35S]Met-tRNA Met f by Sephadex G-100 gel filtration and used as the only source of radioactive methionine in a rabbit reticulocyte in vitro amino acid incorporation system, the transfer of [ 35S]methionine into acid-insoluble material, using A-U-G-(U) x (x = 25 ± 5) as the messenger, was greatly stimulated as compared to the transfer of [ 35S]methionine from free [ 35S]Met-tRNA Met f. Kinetic experiments now confirm that the methionine residue in the ternary complex is a preferred precursor to the N-terminal position of newly initiated polypeptide chains. The formation of the complex is inhibited by low levels of aurintricarboxylate; however, the preformed ternary complex is stable and is not dissociated by the addition of aurintricarboxylate. When the preformed ternary complex is used in the in vitro amino acid incorporation system, aurintricarboxylate inhibits the formation of acid-insoluble material, but only at levels higher than those required for the prevention of ternary complex formation. Thus one reaction in protein biosynthesis which is very sensitive to the presence of aurintricarboxylate is the formation of the ternary complex. This phenomenon may partially explain the action of aurintricarboxylate upon eukaryotic protein synthesis initiation mediated by Met-tRNA Met f.