Initiation of protein synthesis: evidence for messenger RNA-independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit

This paper shows that reticuloeyte lysates contain 40 S/Met-tRNA_f 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-tRNA_f 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-tRNA_f in the 40 S complexes appears to exchange readily with free Met-tRNA_f; when lysates were preincubated with sparsomycin or diphtheria toxin and then incubated with [³⁵S]Met-tRNA_f, the native 40 S subunits were the only ribosomal particles labelled. This experimental system was used to examine whether 40 S/Met-tRNA_f complexes could interact with mRNA; various mRNAs were added shortly after or at the same time as the [³⁵S]Met-tRNA_f. This resulted in a conversion of the 40 S/Met-tRNA_f 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-tRNA_f with the 40 S subunits was not prevented.The 40 S/Met-tRNA_f complexes also participated in initiation on endogenous mRNA, and it was shown that the Met-tRNA_f in this complex was used in preference to free Met-tRNA_f 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-tRNA_f 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-tRNA_f/mRNA initiation complex is formed.     

Partial reaction of peptide initiation inhibited by the reticulocyte hemin-controlled repressor

The hemin-controlled repressor from rabbit reticulocytes inhibits binding of Met-tRNA_f to reticulocyte 40S ribosomal subunits in a partial reaction containing these components, two initiation factor fractions and GTP. The inhibitor does not interfere with the formation of the Met-tRNA_f· initiation factor IF-E₂·GTP complex.     

Studies on native ribosomal subunits from rat liver. Evidence for activities associated with native 40S subunits that affect the interaction with acetylphenylalanyl-tRNA, methionyl-tRNAf, and 60S subunits.

The binding of the initiator tRNA Met-tRNAf, and of acetylphenylalanyl-tRNA, has been examined with rat liver 40S subunits derived from 80S ribosomes by dissociation with native 40S subunits sedimented from the postmicrosomal fraction and with native 40S subunits extracted with high salt-containing solutions. Binding of Met-tRNAf and acetylphenylalanyl-tRNA to derived and to salt-extracted native 40S subunits is observed in the presence of the appropriate polynucleotide template and a highly purified binding factor obtain from the soluble fraction of rat liver homogenates (R.L. IF-1). Native 40S subunits bind acetylphenylalanyl-tRNA in a reaction that requires poly(U) but not exogenous binding factor; however, Met-tRNAf is not bound to native subunits, even when supplemented with the soluble binding factor, or under conditions where factor-independent, high Mg2+-stimulated binding is observed with the derived and the salt-washed native 40S subunits. The extract obtained from native 40S subunits promotes the binding of acetylphenylalanyl-tRNA but not Met-tRNAf to derived and to salt-extracted native subunits. The addition of native 40S extract to incubations containing R.L. IF-1, Met-tRNAf, and derived 40S subunits, inhibits the formation of 40S-Met-tRNAf complex. These data suggest that the binding activity that is specific for 40S subunits and initiator tRNA, and an activity that inhibits the interaction with Met-tRNAf specifically, are both associated with native 40S subunits, and can be extracted from them by treatment with high salt-containing solutions. Derived 40S subunits react quantitatively with 60S particles to form 80S ribosomes which do not bind acetylphenylalanyl-tRNA with binding factor R.L. IF-1. Native 40S subunits react only partly with 60S subunits; about half of the native 40S subunit population forms 80S ribosomes which do not subsequently bind acetylphenylalanyl-tRNA; the remaining native 40S subunits which do not react with 60S particles bind acetylphenylalanyl-tRNA but to a lesser extent. When preformed native 40S-acetylphenylalanyl-tRNA complex is incubated with 60S subunits, about half of the subunits form an 80S-acetylphenylalanyl-tRNA complex, while the rest remains as 40S-acetylphenylalanyl-tRNA. The addition of native 40S subunit salt extract to incubations containing preformed 80S ribosomes dissociates the particles to subunits. These data suggest that in addition to the initiator tRNA binding activity and the activity that inhibits Met-tRNAf interaction, part of the native 40S subunit population also contains an activity that dissociates 80S ribosomes.     

The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells

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-tRNA_f into eIF-2·Met-tRNA_f·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-tRNA_f 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.     

Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf·EIF-1·GTP complex and Met-tRNAf binding to 40S ribosomes

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-tRNA_f·EIF-1·GTP in the presence of Mg⁺⁺ and is also required along with EIF-1 for AUG-directed Met-tRNA_f binding to 40S ribosomes.     

Interaction of rat liver ribosomal subunits with homologous Met-tRNAs

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-tRNA_i to 40 S subunits is greater than that observed with Met-tRNA_m. Also, binding of Met-tRNA_i 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-tRNA_i 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-tRNA_i is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNA^Met) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNA_i binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNA_i 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-tRNA_i is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes.     

Protein synthesis in rabbit reticulocytes XX: A supernatant factor (TDI) inhibits ternary complex (Met-tRNAf·EIF-1·GTP) dissociation and Met-tRNAf binding to 40S ribosomes

A protein synthesis initiation inhibitor, TDI has been partially purified from the reticulocyte cell-supernatant. TDI inhibits the dissociation of the ternary complex, Met-tRNA_f·EIF-1·GTP and also Met-tRNA_f 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.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, 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-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f 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.     

Vaccinia viral core inhibits Met-tRNAf·40S initiation complex formation with physiological mRNAs

Vaccinia viral core inhibits protein synthesis in reticulocyte lysates. In partial reactions using micrococcal nuclease treated reticulocyte lysates, the viral core inhibits Met-tRNA_f 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-tRNA_f 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-tRNA_f·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.     

A functional interaction between methionyl-transfer RNA hydrolase and a transfer RNA binding factor

Met-tRNA_f bound at low Mg ion concentrations to rabbit reticulocyte 40 S ribosomal subunits in the presence of ApUpG and a eukaryotic tRNA binding factor serves readily as a substrate for a Met-tRNA hydrolase from rabbit reticulocytes. This hydrolysis occurs rapidly at 0 °C, appears to be specific for Met-tRNA_f, and is not inhibited by 60 S ribosomal subunits. These reactions may be responsible for the accumulation of deacylated tRNA_f^Met observed in ribosomes isolated from sodium fluoride-treated cells.     

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.     

A rapid and sensitive assay for the detection of eukaryotic ribosome dissociation factors.

A rapid and sensitive assay has been developed for the factor-dependent dissociation of eukaryotic ribosomes. This assay takes advantage of the observation that initiation factor eIF-2 will bind Met-tRNA_f^met to 40 S subunits but not to 80 S ribosomes. Incubation of wheat germ ribosomes at 1 mm Mg²⁺ results in their dissociation into 40 S subunits. These subunits spontaneously reassociate when the Mg²⁺ concentration is raised to 4 mm. However, if the incubation at 1 mm Mg²⁺ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion of the subunits will fail to reassociate when the Mg²⁺ concentration is raised to 4 mm. The 40 S subunits remaining due to the presence of the dissociation factor can bind [³⁵S]Met-tRNA_f^met in the presence of wheat germ eIF-2. The [³⁵S]Met-tRNA_f^met bound to the 40 S subunits is readily detected by its retention on a Millipore filter.     

Inhibition of exogenous RNA-dependent protein synthesis by a low-molecular-weight RNA from nuclear ribonucleoprotein particles of adenovirus-infected HeLa cells

Low-molecular-weight RNA (4S to > 5.5S) isolated from nuclear ribonucleo-protein particles of adenovirus-infected HeLa cells inhibited cell-free protein synthesis directed by polyribosomal RNA from rabbit reticulocytes by more than 80%. In a reconstituted system inhibitory RNA did not prevent the binding of Met-tRNA_f-GTP-IF ternary complex to 40S subunits; however, it repressed the formation of 80S from 40S-mRNA complex and 60S subunits. In binding assays in which authentic IF-M2A and IF-M2B were present, the inhibitor competed with messenger molecules for binding site(s) in IF-M2B. The inhibitory RNA appears to be a 5.5S RNA.     

Temperature sensitivity of protein synthesis initiation. Inactivation of a ribosomal factor by an inhibitor formed at elevated temperatures.

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-tRNA_f 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-tRNA_f 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-tRNA_f 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.     

Characterization of a native mRNA containing preinitiation complex from rabbit reticulocytes: RNA and protein constituents

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-tRNA_f·GTP] complex which may occur in vivo as a translatable intermediate in the initiation sequence.     

Factor-independent interaction of met-tRNA i and ApUpG with 40 S subunits of rat liver ribosomes

The small (40 S) subunit of rat liver ribosomes is capable of binding the initiator tRNA (Met-tRNA_i), in the absence of added protein factors, in marked preference to other aminoacyl-tRNAs. This binding requires magnesium ions, is codon (ApUpG)-specific, is not obtained with 60 S subunits, and is significantly higher than that observed with 80 S ribosomes. The 40 S subunit also exhibits a preference for ApUpG over several other trinucleotides. The reaction is inhibited by 60 S particles; it is also inhibited by compounds that effect chain initiation such as edeine and aurintricarboxylic acid, but not by cycloheximide, tetracycline or KF. All other aminoacyl-tRNAs, including Met-tRNA_m, bind more efficiently to 80 S ribosomes at low MgCl₂ concentrations with EF1 or in high Mg⁺⁺-containing solutions.     

Formation of an Initiation Complex with Purified Mammalian Ribosomal Subunits

PROTEIN synthesis in at least some mammalian cells is probably initiated by Met-tRNA_f^1–3, which binds to salt-washed ribosomes at low Mg²⁺ concentrations in the presence of AUG and initiation factors^4,5. Myosin mRNA will bind to 40S ribosomal subunits and if this represents a true initiation complex, it should bind specific initiator tRNA^6,7. We report that an initiation complex specific for Met-tRNA_f can be formed with the 40S ribosomal subunit isolated from mouse plasmacytoma tumours.     

Protein synthesis in rabbit reticulocytes XXII+: a heat stable dialyzable factor (EIF-I*) modulates Met-tRNAf binding to EIF-1.

The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNA_f·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-tRNA_f binding activity of EIF-1 in the presence of 1 mM Mg⁺⁺; in the presence of EIF-1¹, Met-tRNA_f 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-tRNA_f binding to EIF-1 in the absence of Mg⁺⁺ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system.     

Protein synthesis in rabbit reticulocytes. XIII. Lack of mRNA (poly r (A)) binding activity in highly purified EIF-1.

Met-tRNA_f^Met binding factor (EIF-1) has been purified more than 100 fold over crude high salt (0.5 M KCl) ribosomal wash. The purified factor binds 2 nmoles Met-tRNA_f^Met per mg protein and shows very little poly r(A) binding activity. Crude ribosomal high salt wash possesses significant amounts of poly r(A) binding activity and also binds to other RNAs. The bulk of this unspecific RNA binding protein is separated from EIF-1 by DEAE-cellulose chromatography.     

Mutually exclusive binding of messenger RNA and initiator methionyl transfer RNA to eukaryotic initiation factor 2

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-tRNA_f. 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-tRNA_f and GTP, but cannot do both at once.