Formation Of 80S Initiation Complex Research Articles

Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.

Investigation of the role of uncharged tRNA in the regulation of polypeptide chain initiation by amino acid starvation in cultured mammalian cells; a reappraisal.

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40‐S subunit. Met‐tRNAf ribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells. Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine‐starved cells. In cell‐free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40‐S initiation complex formation nevertheless remains defective after lysine starvation of the cells. Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell‐free extracts inhibits neither 40‐S initiation complex formation nor overall protein synthesis in vitro. Addition of lysine to an extract from lysine‐starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40‐S initiation complex formation or protein synthesis in vitro. These results suggest that amino acid starvation regulates the formation of 40‐S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.

The effects of haem on translational control of protein synthesis in cell-free extracts from fed and lysine-derived Ehrlich ascites tumour cells.

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit X Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.

Vaccinia viral core inhibits Met-tRNA f·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.

Effect of oligonucleotide AGAGGAGGU on protein synthesis in vitro

The oligonucleotide AGAGGAGGU, complementary to the 3' end of 16S RNA has been shown to inhibit 70S initiation complex formation on E. coli ribosomes (Taniguchi, T. and Weissmann, C., 1978. Nature, 275, 770-772). We have prepared this nonanucleotide in larger quantities by a combination of DEAE cellulose-urea chromatography and reverse phase (RPC 5) chromatography. The inhibitory effect of AGAGGAGGU on initiation complex formation has been confirmed. Furthermore, when added to a complete system for in vitro protein synthesis, the translation of Q beta RNA was inhibited by the nonanucleotide. No selectivity was observed in the inhibition of the coat protein and replicase protein synthesis. When both Q beta RNA and pAUG were present, some stimulation of pAUG binding to 70S ribosomes was observed on addition of AGAGGAGGU, as previously reported (Taniguchi and Weissmann, ibid). No effect was observed in the absence of Q beta RNA. This observation is discussed.

The mechanism of action of eukaryotic initiation factor 4C in protein synthesis.

Binding of eukaryotic initiation factor 4C (eIF-4C) to a 40-S initiation complex could be detected by analysis of assay mixtures for 40-S initiation complex formation on columns of Sepharose 6B and on sucrose gradients. On Sepharose 6B columns an almost stoichiometric binding of [14C]eIF-4C was observed, provided that eIF-1, eIF-2, eIF-3, Met-tRNAf and mRNA were included in the assay mixtures. On sucrose gradients the presence of 0.5 microM edeine was an additional prerequisite for stable binding of eIF-4C. The presence of eIF-4C on the 40-S initiation complex resulted in a pronounced increase of its stability, which might explain the observed stimulatory effect of the factor on the binding of Met-tRNAf into initiation complexes. The interaction between eIF-4C and the small ribosomal subunit presumably occurs at an early stage of the initiation process, since eIF-4C was capable of dissociating 80-S ribosomes into subunits. Protein synthesis in a 'pH-5' assay was almost totally dependent on the addition of eIF-4C, when the assay was performed with purified, nearly homogeneous, initiation factors.

Stimulation of initiation factor eIF-2 by a rat liver protein with GDPase activity

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-tRNA f] 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.

Inhibition of 80S Initiation Complex Formation by Infection with Poliovirus

Anisomycin has been shown to stabilize ribosome initiation complexes containing messenger RNA and met-tRNAf met to high salt conditions. Extracts from HeLa cells treated with 5 X 10(-7) M-anisomycin for 15 min accumulate 80S initiation complexes which can be detected by their absorbance in sucrose gradients. Poliovirus-infected cells fail to form the 80S initiation complex early after infection, when inhibition of host cell protein synthesis occurs. These complexes re-form later in infection after virus RNA is synthesized. No re-formation occurs in the absence of virus replication. Thus, the step in protein synthesis inhibited by poliovirus precedes the entry of components into the 80S initiation complex.

Initiation Factor IF-3 and the Binary Complex between Initiation Factor IF-2 and Formylmethionyl-tRNA Are Mutually Exclusive on the 30-S Ribosomal Subunit

The formation of 30-S initiation complexes depends strongly on initiation factor IF-3; at molar ratios of IF-3 to 30-S ribosomes up to one a stimulation is observed, whereas at ratios higher than one, initiation complex formation declines strongly. The target of the observed inhibition of fMet-tRNA binding at high concentrations of IF-3 is the 30-S initiation complex itself. On the one hand addition of IF-3 to preformed 30-S initiation complexes leads to a release of bound fMet-tRNA which is linear with the amount of factor added, whereas no effect on isolated 70-S initiation complexes is seen. The release of fMet-tRNA from preformed 30-S initiation complexes is accompanied by a release of IF-2 in a one-to-one molar ratio which is in agreement with our previous findings showing that binding of fMet-tRNA takes place via a binary complex: IF-2 . fMet-tRNA (Eur. J. Biochem. 66, 181--192 and 77, 69--75). On the other hand increasing amounts of both IF-2 and fMet-tRNA relieve the IF-3-induced inhibition of 30-S initiation complex formation. From these findings it is concluded that IF-3 and the IF-2 . fMet-tRNA complex are mutually exclusive on the 30-S ribosome. This implies that under our experimental conditions MS2 RNA binding precedes fMet-tRNA binding if one accepts that the presence of IF-3 on the 30-S subunit is obligatory for messenger binding.

Consequences of a Specific Cleavage in situ of 16-S Robosomal RNA for Polypeptide Chain Initiation

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with our previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A. 73, 702--706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can be compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subunit interaction.

Identification of features in 5′ terminal fragments from reovirus mRNA which are important for ribosome binding

Four types of experiments were carried out with reovirus messenger RNAs or with 5′ terminal fragments of known sequence to identify features in mRNA which appear to be important for formation of initiation complexes with ribosomes. With a number of reovirus mRNAs, 40S initiation complexes had been previously shown to protect a significantly larger segment of the RNA (including the 5′ terminal m7G) than that protected by 80S initiation complexes. Each 80S-protected sequence had an AUG codon and was a subset of the 40S-protected sequence from the same message. When 40S- and 80S-protected fragments were tested for ability to rebind to ribosomes, the 80S-protected fragments showed considerably lower binding ability, implying that the “extra” sequences protected by 40S initiation complexes contribute to ribosome attachment. Nevertheless, wheat germ ribosomes select the same 5′ terminal initiation site in each reovirus mRNA, irrespective of the presence or absence of m7G on the message. This was demonstrated by comparing fingerprints of the ribosome-protected regions obtained with methylated versus unmethylated RNA. The contribution of m7G to formation of initiation complexes is therefore quantitative rather than qualitative. Limited T1 RNAase digestion of isolated 5′ terminal fragments from several reovirus messages generated a series of smaller fragments which were analyzed for ability to rebind to ribosomes. Partial digestion products up to 30 nucleotides in length which retained the 5′ cap but not the AUG codon were unable to associate stably with ribosomes, whereas every AUG-containing fragment that was analyzed was able to form initiation complexes. The efficiency of binding of certain AUG-containing fragments, however, was reduced by removal of either the 5′ terminal region, including the cap, or of sequences comprising the beginning of the coding region, on the 3′ side of the AUG. Complex formation between messenger RNA and ribosomes was inhibited by the trinucleotide AUG, but not by various other oligonucleotides. Although the inhibition was specific, a vast excess of trinucleotide was required for moderate inhibition of 80S complex formation, and the same concentration of AUG failed to inhibit formation of 40S initiation complexes.

Nucleic acid helix-unwinding properties of ribosomal protein S1 and the role of S1 in mRNA binding to ribosomes.

The presence of ribosomal protein S1 in 30S ribosomes is indispensable for the formation of 30S initiation complexes with natural mRNA. The 30S subunits lacking S1 retain activity with AUG as mRNA and are also active in poly(rU)-directed binding of Phe-tRNA. Isolated protein S1 stoichiometrically disrupts the secondary structure of helical and stacked single-stranded polynucleotides and converts them into their fully or partially denatured forms. A mono-N-ethylmaleimide derivatives of S1 is nearly devoid of any RNA helix-unwinding properties but is readily incorporated into 30S subunits deficient in S1. The resulting N-ethylmaleimide-S1-containing 30S subunits are completely inactive in the binding of MS2 [3H]RNA and in the formation of an initiation complex with MS2 RNA as mRNA. They retain activity in the binding of the initiator fMet-tRNA in response to the trinucleotide AUG and in the binding of Phe-tRNA in response to poly(U). They also retain the capacity to bind 50S subunits and to form 70S couples. These results suggest that a correlation exists between the RNA helix-unwinding capacity of isolated S1 and the function of S1 in the ribosomal binding of natural mRNA when the protein becomes part of the 30S subunit.

The Role of Complexes between the 40-S Ribosomal Subunit and Met-tRNAMetf in the Initiation of Protein Synthesis in the Wheat-Germ System

We have demonstrated the formation of a complex between the 40-S ribosomal subunit and Met-tRNAMetf in the absence of mRNA in an unfractionated wheat-germ system. Two types of experiment indicate that these complexes (40S · Met-tRNAMetf) are true intermediates in the formation of 80-S initiation complexes containing mRNA, Met-tRNAMetf and both ribosomal subunits. Firstly, isolated 40S · Met-tRNAMetf complexes were used preferentially over free Met-tRNAMetf in the formation of 80-S initiation complexes on addition of mRNA. Secondly by the use of methioninol-AMP, an inhibitor of methionyl-tRNA synthetase, to deplete endogenous Met-tRNAMetf pools, we showed that the binding of mRNA to ribosomes was strictly dependent on the presence of Met-tRNAMetf. The 40S · Met-tRNAMetf complexes had other properties consistent with their role as intermediates in initiation. The radioactivity in 40S · Met-tRNAMetf complexes labelled with [35S]Met-tRNAMetf was shifted into 80-S initiation complexes on addition of mRNA. This shift was prevented by aurintricarboxylic acid, which inhibits the binding of mRNA to ribosomes. In the presence of edeine, which blocks the binding of the 60-S subunit, the radioactivity was found in complexes containing mRNA, Met-tRNAMetf and 40-S subunits. These results were confirmed by the use of labelled mRNA. We propose that the mechanism of protein chain initiation is universal to all cells, the first step being the formation of a complex between the small ribosomal subunit and (f)Met-tRNAMetf. This complex can then bind mRNA in a process which must to some extent involve selection of the initiator AUG codon through interactions with the anticodon of the (f)Met-tRNAMetf. The binding of mRNA is followed by the addition of the large ribosomal subunit and the formation of an initiation complex capable of forming the first peptide bond.

The mechanism of inhibition of initiation of protein synthesis in reticulocyte lysates by pyrocatechol violet

1. Pyrocatechol violet inhibits the initiation of protein synthesis in reticulocyte lysates. It prevents the formation of 80-S initiation complexes by inhibiting the binding of mRNA to 40-S initiation complexes. 2. Pyrocatechol violet inhibits the hydrolysis of methionyl-tRNA met f by reticulocyte lysates or reticulocyte cell sap. 3. Concentrations of pyrocatechol violet sufficient to inhibit initiation have little effect on amino acid activation. Higher concentrations of pyrocatechol violet were strongly inhibitory.

Cooperative effects of initiation factors and fMet-tRNA in the formation of the 40-S initiation complex.

In this paper the mode of action of IF-1 in 40-S initiation complex formation was studied with MS2 RNA as messenger. Using initiation factors IF-2 and IF-3 labeled in vitro it appeared that IF-1 did not influence the binding of these factors in the absence of fMet-tRNA. However, in the presence of fMet-tRNA it was found that the enhancement of the fMet-tRNA binding by IF-1 was accompanied with an equimolar increase in binding of IF-2. Moreover, it appeared that also in absence of IF-1, fMet-tRNA binding is coupled with an equimolar enhancement of the IF-2 binding, which suggests the existence of a preribosomal complex between IF-2 and fMet-tRNA. The apparent Km values for both the binding of fMet-tRNA and IF-2 to 30-S subunits were determined and appeared to be equal, which makes a functioning of such a preribosomal complex in protein initiation very likely. The participation of GTP in this complex will be discussed. Functions of IF-1 in dissociation and recycling of IF-2, described by others, and the stimulation on the 30-S subunit level might well be explained as pleiotropic effects of one basic action of IF-1, i.e. a conformational change of 30-S subunits.

Chapter 4 Regulation of Messenger RNA Translation During Insect Development

Insect metamorphosis is the differentiation at the postembryonic level. The process of cell differentiation is linked to the mechanisms that direct specific protein synthesis rather than with the ultimate appearance of protein molecules. The development of pupa into adult occurs in a differentiating system, where many changes take place, usually in one direction. In insects, growth and differentiation are regulated by several hormones. The use of juvenile hormone helps to study the mechanisms of gene expression. The major influence of juvenile hormone in promoting the synthesis of the two classes of cuticular protein-adult and pupal is at the translational level. This is facilitated by controlling the translation of cuticular messenger ribonucleic acid (mRNA). The translational control is mediated by the appearance of new rate-limiting aminoacyl-transfer ribonucleic acid (tRNA). The chapter explains the steps involved in protein synthesis. One of the characteristics of the differentiated cell is its ability, at one point in its development, to synthesize a large amount of a specific protein. This is accomplished by the production of stable mRNA. If a specific mRNA is stable, this amounts to the amplification of this specific gene. The tRNA and its activating enzymes are the limiting factors in the microsomal cell-free system for the regulation of cuticular mRNA translation. The chapter discusses the regulation of cuticular mRNA translation in cell-free systems from Tenebrio pupae . In addition, the chapter discusses the experiments on the initiation of mRNA translation during the development of Tenebrio pupae . In eukaryotic organisms, protein synthesis is initiated in a manner similar to the bacterial protein synthesis. The specificity in the formation of 80S initiation complex suggests that mRNA contains, besides the AUG codon for methionine, a specific sequence of nucleotides that is recognized by some elements of the initiation machinery.

Effect of lincomycin and clindamycin on peptide chain initiation.

Lincomycin does not affect initiation factor-dependent formation of 70S initiation complexes formed with fmet-tRNA(F), the initiation triplet A-U-G, and 70S ribosomes, whereas its 7-chloro-derivative clindamycin substantially stimulates this process. Conversely, lincomycin stimulates nonenzymatic formation of the 70S complex, but clindamycin does not. Both antibiotics stimulate the assembly of non-enzymatically formed 70S initiation complexes with R(17) phage ribonucleic acid and exert little effect on those formed in the presence of initiation factors. The formation of 30S initiation complexes is stimulated or remains unaffected by lincomycin or clindamycin except when initiation occurs in the presence of very low Mg(2+) concentrations. In this case, both antibiotics inhibit the assembly of the 30S complexes regardless of the messenger present.

Thiostrepton inhibition of initiation factor 1 activity in polypeptide chain initiation in Escherichia coli.

Thiostrepton, a peptide antibiotic, inhibits the GTP-dependent 70S initiation complex formation (as measured by binding of fMet-tRNA to ribosomes and concomitant hydrolysis of GTP) only when initiation factor 1 is present to permit catalytic recycling of initiation factor 2 in the initiation reaction. When initiation factor 1 is absent, the binding of fMet-tRNA and GTP hydrolysis occur stoichiometrically with respect to initiation factor 2, and thiostrepton has no effect on either reaction under these conditions. Detailed analysis of this inhibition process shows that thiostrepton prevents catalytic recycling of initiation factor 2 by blocking the action of initiation factor 1, which is required for the dissociation of initiation factor 2 from the 70S initiation complex. This dissociation is necessary for the catalytic reutilization of initiation factor 2 in the initiation reaction. The antibiotic does not directly inhibit GTP hydrolysis per se in initiation. The inhibition of fMet-tRNA binding to ribosomes by thiostrepton is also dependent on the concentration of GTP; the inhibition is most pronounced at low concentrations of GTP, but at a high molar ratio of GTP to thiostrepton, the inhibition is completely abolished.

Reversal of pactamycin inhibition of methionyl-puromycin synthesis and 80S initiation complex formation by a ribosomal joining factor.

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA(f) to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type. In the absence of the factor, [(35)S]Met-puromycin was not synthesized and [(35)S]Met-tRNA(f) bound only to the small ribosomal subunit. The amount of [(35)S]Met-tRNA(f) bound to 80S ribosomes bearing endogenous mRNA and the amount of [(35)S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a "joining factor," and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.

The effects of initiation factor 3 on the formation of 30S initiation complexes with synthetic and natural messengers

Initiation factor IF-3 is required for the binding of fMet-tRNA to 70S ribosomes directed by AUG, poly (U,G), f 2RNA and T 4 late RNA as well as for the binding of acPhe-tRNA directed by poly (U). In contrast, IF-3 is not required for the binding of the initiator aminoacyl-tRNAs to isolated 30S subunits directed by the synthetic messengers, but is required for maximal formation of initiation complexes with natural messengers. These data indicate that with synthetic messengers the sole function of IF-3 is to dissociate the 70S ribosomes into subunits, whereas with natural messengers IF-3 is required not only for dissociation of the ribosomes but also for the binding of the messenger to the 30S subunit.