Activity of partially purified protein chain initiation factors from the livers of dimethylnitrosamine-treated rats

Partially purified polypeptide chain initiation factors were prepared from the 0.5 M KCl wash of rat liver microsomes. Their activities in connection with dimethylnitrosamine (DMNA)-induced inhibition of protein synthesis were studied by use of the following reactions: (1) poly(U)-directed binding of Phe-tRNA to ribosomes, (2) formation of a GTP-dependent ternary initiation complex with Met-tRNAf, (3) binding of Met-tRNAf to 40-S ribosomal subunits, (4) assembly of a Met-tRNAf containing 80-S ribosomal initiation complex and (5) ribosome-dependent GTPase activity. The inhibition of protein synthesis with DMNA was not associated with a loss of factor activity in any of these reactions. In the binding of Met-tRNAf to 40-S subunits there was a noticeable increase, probably related to the stability of the resulting complex. The Met-tRNA deacylase activity was also increased.     

Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S.AUG.Met.tRNAf.eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2.GDP binary complex and Pi formed were released from the ribosomal complex whereas Met-tRNAf remained bound to 40 S ribosomes as a Met-tRNAf.40 S.AUG complex. Reactions carried out with biologically active 32P-labeled eIF-5 indicated that this protein was not associated with the 40 S.AUG.Met-tRNAf complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S.AUG.Met-RNAf complex, free of eIF-2.GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNAf into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S.AUG.Met-tRNAf and dissociation of the eIF-2.GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S.AUG.Met-tRNAf complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions. (40 S.AUG.Met-tRNAf.eIF-2.GTP) eIF-5----(40 S.AUG.Met-tRNAf) + (eIF-2.GDP) + Pi (1) (40 S.AUG.Met-tRNAf) + 60 S----(80 S.AUG.Met-tRNAf) (2) 80 S initiation complex.     

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).     

Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition.

The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome-mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5' to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that involve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor-mediated stimulation of complex formation by a combination of eIF-4A, -4B, and -4F was observed, and was one which required the presence of ATP. This factor-mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5'-p-fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested.     

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.     

Reassociation of eukaryotic ribosomal subunits: dependence of reassociation on formation of a 40S initiation complex: effects of aurintricarboxylic acid and pactamycin

Aurintricarboxylic acid and pactamycin inhibited initiation factor catalyzed reassociation of ribosomal subunits to form 80S couples and subsequent polyphenylalanine synthesis although their effects were qualitatively different. The two inhibitors prevented the formation of 80S monomers if they were present with 40S subunits in the reassociation mixture before addition of large subunits; they did not inhibit protein synthesis nor reassociation if they were added with the 60S subunits after formation of a small subunit initiation complex. Thus creation of a 40S initiation complex precedes addition of the large subunit and formation of an 80S monomer. An additional finding was that aurintricarboxylic acid preferentially inhibited the formation of inactive 40S–60S couples.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.     

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.     

Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

Stability of protein synthesis initiation complexes in the presence of edeine

The antibiotic inhibitor edeine has been used to study various aspects of the initiation of protein synthesis in a rabbit reticulocyte cell-free system. Edeine prevents assembly of the 80S initiation complex while allowing accumulation of a 44S initiation intermediate. The complete 80S initiation complex, once formed, is stable in the presence of edeine. The functioning of the initiation complex, as judged by release of methionyl-puromycin, is only partially inhibited by a concentration of edeine which fully inhibits formation of the initiation complex. The above effects of edeine on a eukaryotic system differ from the effects edeine has been found to have in a prokaryotic system.     

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.     

The use of glutaraldehyde fixation to analyze the mechanism of factor catalyzed reassociation of eukaryotic ribosomal subunits

Glutaraldehyde fixation was used to analyze the mechanism of reassociation of ribosomal subunits catalyzed by a factor in rat liver cytosol. Unstable 40S–60S couples formed spontaneously in buffer alone; the couples were dissociated by hydrostatic pressure during centrifugation unless they were fixed with glutaraldehyde. Increased numbers of stable 80S ribosomes were formed in the presence of poly (U), Phe-tRNA and G-25 fraction (which contains the initiation factor EIF-1). The factor would seem then to both increase formation of 80S ribosomes and stabilize the monomer. An additional effect of the factor is to inhibit the formation of the unstable 40S–60S couples which form in the presence of Phe-tRNA alone.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

Stimulation of enzymatic phe-tRNA binding to mammalian ribosomes by thallium ions at concentrations blocking other ribosomal functions.

Thallium acetate (TIOAc) effectively stimulates poly(U)-directed Phe-tRNA binding to mouse ascitic tumour ribosomes under conditions when other ribosomal functions are completely blocked. The TI+ optimum is about 200 mM. The reaction is stimulated by EF-1, but not significantly by GTP. EF-1-dependent ribosomal GTPase is inhibited by T1+. The isolated Phe-tRNA . ribosome complex is relatively stable. The bound Phe-tRNA does not react with puromycin in the presence of 175 mM KCl. The complex formed in the presence of 90-100 mM TlOAc can, after isolation, be directly utilized for polyphenylalanine synthesis. The complex formed at 200 mM TlOAc is less active, apparently because of damage to the 60-S subunits. TlOAc at low concentrations (8 mM) stimulates K+ -containing poly(U)-translating systems, probably by stabilizing the translation complex.     

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.     

Influence of heating on the formation of ribosome couples and 80S initiation complex from the 40S and 60S ribosomal subunits from rabbit reticulocytes*

Summary Poly(U)-and AUG-dependent initiations were studied under conditions in which the ribosomal subunits from rabbit reticulocytes form 80S ribosome couples. The AUG-dependent initiation took place only in the presence of 40S subunits and not in that of 80S ribosome couples. The poly(U)-dependent initiation could take place on 80S ribosome couples as well as on 40S subunits. Heating of 60S subunits removed their inhibitory effect on AUG-dependent initiation without affecting their ability to attach to the 40S-AUG-initiator-tRNA complex and covert it to the more thermostable 40S-AUG-60S complex. This complex could bind initiator-tRNA reversibly. Two components seemed to be present in ribosomal high salt wash which antagonistically caused the disappearance and persistence of the inhibitory effect of 60S subunits on AUG-dependent initiation. Paper No. 3 on “Studies on Rabbit Reticulocytc Ribosomes”. Preceding paper is by Chatterjce, S.K., Kazemie, M., Matthaei, H., noppe-Seyler’s Z. Physiol. Chem.345, 481 (1973).     

Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.     

Structure of a novel flavin chromophore from Avena coleoptiles, the possible ''blue light'' photoreceptor

The effect of edeine on the translation of mRNA or poly(U)-directed polyphenylalanine synthesis has been studied in an edeine-resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal-nuclease-treated lysate, and in a high-salt-treated lysate. The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates; these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA. It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild-type subunits. Since the mRNA binding is inhibited by 7-methylguanosine 5'-monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA. The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited. However, edeine inhibition of [3H]methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA. The results indicate that the mutant may carry a specific change in a messenger-binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger-ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.     

Nature of eukaryotic proteins required for joining of 40S and 60S ribosomal subunits

A mixture of 40S and 60S subunits from salt-washed rabbit reticulocyte ribosomes fails to promote methionyl-puromycin synthesis under conditions in which an AUG-40S-Met-tRNA_i initiation complex, but not an 80S complex, is readily formed. This suggests that the inability of the system to form methionyl-puromycin is due to failure of the subunits to join. When $\text{Artemia salina}$ 60S subunits are substituted for their reticulocyte counterparts, the resulting hybrid system readily forms an 80S initiation complex and synthesizes methionyl-puromycin. Activity of the reticulocyte 60S subunits can be restored by factors IF-M2A and IF-M2B. This suggests that one or both of these factors may be 60S proteins, essential for subunit joining, that may be removed from ribosomes by salt washing procedures.