The involvement of a complex between formylmethionyl-tRNA and initiation factor IF-2 in prokaryotic initiation.

A complex between initiation factor IF-2 and fMet-tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde. The IF-2 - FMet-tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di of triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF-2 - fMet-tRNA complex formation is not influenced by initiation factors IF-1 and IF-3. The binary complex can be bound to the 30-S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF-2 - fMet-tRNA complex and the nucleotide moiety to the 30-S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF-2 - fMet-tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate had no effect.     

Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli.

1. Studies on the function of initiation factor 1 (IF-1) in the formation of 30 S initiation complexes have been carried out. IF-1 appears to prevent the dissociation of initiation factor 2 (IF-2) from the 30 S initiation complex. The factor has no effect on either the initial binding of IF-2 nor does it increase the amount of IF-2 dependent fMet-tRNA and GTP bound to the 30 S subunit. Bound fMet-tRNA remains stable to sucrose gradient centrifugation even in the absence of IF-1. 2. It is postulated that the presence of IF-2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S-dependent GTPase activity of IF-2 can hydrolyze GTP. This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer. In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF-1 could be depleted of their complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet-tRNA into peptide linkages. These results indicate that 30 S complexes lacking GTP do not require IF-2 for formation of active 70 S complexes. 3. IF-1, which is required to prevent dissociation of IF-2 from the 30 S initiation complex, is also required for release of IF-2 from ribosomes following 70 S initiation complex formation. The mechanisms of the release of IF-2 has been studied in greater detail. Evidence is presented which rules out the presence of a stable IF-2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF-1 and guanine nucleotides necessary for effecting the release of IF-2. IF-2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF-1.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.     

Specificity and properties of the destabilization, induced by initiation factor IF-3, of ternary complexes of the 30-S ribosomal subunit, aminoacyl-tRNA and polynucleotides.

Initiation factor IF-3 causes the destabilization of preformed ternary complexes of 30-S ribosomal subunit, codons and aminoacyl-tRNAs or peptidyl-tRNA. This destabilization is dilution-dependent and affects all ternary complexes with the exception of those containing the initiator fMet-tRNA, which remain more resistant to IF-3-induced destabilization under the various conditions studied. Several possible reasons for this specificity have been examined. It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing fMet-tRNA, (b) the amoung of aminoacyl-tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group. On the other hand, the nature of the polynucleotide in response to which the ternary complex is formed was found to influence the amount of aminoacyl-tRan bound to the ribosome, and to some extent the amount of aminoacyl-tRNA which can be relased. The ternary complex containing the mischarged initiator tRNA fVal-tRNAfMet displays greater resistance to the IF-3-induced destabilization than the complex containing fVal-tRNAVal. These results indicate that the specificity of the IF-3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide. The IF-3-induced destabilization of ternary complexes was found to be little affected by variations in reaction conditons, so that this IF-3 activity can be used to measure the stoichiometric binding of IF-3 to the ribosome over a broad range of pH and K+ and Mg2+ concentrations. Several antibiotics have been tested for their capacity to interfere with this reaction; only high concentrations of tetracycline blocked this IF-3 activity.     

Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.     

Participation of initiation factor IF-3 in the binding of AcPhe-tRNA to the 30S ribosomal subunit

Initiation factor IF-3 is required in addition to IF-1 and IF-2 for maximal initial rate of poly(U)-directed binding of AcPhe-tRNA to 30S ribosomal subunits of $\text{E. coli}$. Incubation periods longer than 10 sec, by which time the reaction is virtually over, progressively obscure the requirement for IF-3 in AcPhe-tRNA binding. IF-3 also stimulates the poly(A, G, U)-directed binding of fMet-tRNA to the 30S ribosomal subunit, but in this case, significant stimulation can still be observed even with extended incubation. These results indicate that IF-3 functions similarly in the translation of synthetic mRNA, as it does with natural mRNA, participating in ribosome dissociation and in the formation of the initiation complex from the 30S ribosomal subunit.     

Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes

The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates. Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex.     

A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA _inff ^supMet . Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA _inff ^supMet . Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU₃ as mRNA, elongator-tRNAs competed with fMet-fRNA _inff ^supMet and with tRNA _inff ^supMet . fMet-tRNA _inff ^supMet reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU₃ complex at the A site only if fMet-tRNA _inff ^supMet or tRNA _inff ^supMet filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.     

Identification and characterization of large, complex forms of chloroplast translational initiation factor 2 from Euglena gracilis

Chromatography of partially purified preparations of Euglena gracilis chloroplast initiation factor 2 (IF-2chl) on gel filtration resins indicates that this factor is present in high molecular mass forms ranging from 200 to 700 kDa. The higher molecular weight complexes can be separated from the 200,000 Mr form of this factor by chromatography on DEAE-cellulose. Further purification indicates that the majority of the IF-2chl is present as dimeric, tetrameric, and probably hexameric complexes of polypeptides of 97,000-110,000 in molecular weight. In addition, one form consisting of subunits of about 200,000 Mr has been detected. All of these species are active in promoting fMet-tRNA binding to chloroplast 30 S subunits in a message-dependent reaction. Initiation complex formation promoted by IF-2chl requires the presence of GTP. Similar levels of binding are obtained when GTP is replaced by a nonhydrolyzable analog suggesting that IF-2chl is acting stoichiometrically rather than catalytically under the conditions used. The activity of this factor is stimulated by the presence of either Escherichia coli or chloroplast IF-3. None of the forms of IF-2chl detected is active on E. coli ribosomes.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.     

Initiation factor 3 requirement for the formation of initiation complexes with synthetic oligonucleotides

Initiation factor IF-3 is required for the poly (U)-directed binding of N-acetyl-Phe-tRNA to 70S ribosomes as well as for the binding of fMet-tRNA directed by poly (U,G), AUG, and bacteriophage f₂ RNA. The formation of the 70S initiation complex is dependent upon IF-2 and is stimulated by IF-1. The requirement for IF-3 is not alleviated by high concentrations of the synthetic templates.     

Consequences of a specific cleavage in situ of 16-S ribosomal 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.     

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), f₂RNA and T₄ 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.     

Initiation of protein synthesis in animal mitochondria. Purification and characterization of translational initiation factor 2

Bovine liver mitochondrial translational initiation factor 2 (IF-2mt) has been purified to near homogeneity. The scheme developed results in a 24,000-fold purification of the factor with about 26% recovery of activity. SDS-polyacrylamide gel electrophoresis indicates that IF-2mt has a subunit molecular mass of 85 kDa. IF-2mt promotes the binding of formyl(f)Met-tRNA to mitochondrial ribosomes but is inactive with the nonformylated derivative. IF-2mt is active on chloroplast 30 S ribosomal subunits, but IF-2chl has no activity in promoting fMet-tRNA binding to animal mitochondrial ribosomes. IF-2mt is sensitive to elevated temperatures and is inactivated by treatment with N-ethylmaleimide. It is partially protected from heat and N-ethylmaleimide inactivation by the presence of either GTP or GDP suggesting that guanine nucleotides may bind to this factor directly. The binding of fMet-tRNA to mitochondrial ribosomes requires the presence of GTP and is inhibited by GDP. DeoxyGTP is very effective in replacing GTP in promoting fMet-tRNA binding to ribosomes and some activity is also observed with ITP. No activity is observed with ATP, CTP, or UTP. Nonhydrolyzable analogs of GTP can promote formation of both 28 S and 55 S initiation complexes indicating that GTP hydrolysis is not required for subunit joining in the animal mitochondrial system.     

Circular dichroism analysis of the ribosomal binding of initiation factor IF-3

The circular dichroism spectra of Escherichia coli 30 S ribosomal subunits have been determined between 200 and 320 nm in the presence and in the absence of initiation factor IF-3. The addition of IF-3 did not produce any major alteration of the circular dichroism spectrum of the 30 S subunits between 320 and 240 nm, but resulted in an increase of the negative ellipticity between 240 and 205 nm. The effect was maximal for an IF-3:30 S molar ratio of approximately one, and further addition of IF-3 did not lead to a further increase of ellipticity. A similar effect was not seen when the 30 S ribosomal subunits were previously heat-inactivated to destroy their IF-3 binding capacity. These data indicate that the ribosomal binding of IF-3 may be accompanied by an increase in the secondary structure of the ribosomal proteins, but does not involve any major net change in the secondary structure of the rRNA.     

The effect of initiation factor IF-1 on the dissociation of 70-S ribosomes of Escherichia coli.

By means of exchange studies, in which 3H-labelled 50-S subunits and unlabelled 70-S ribosomes from Escherichia coli MRE 600 were used, it has been demonstrated that the 30-S subunit is the only target for IF-3 in the dissociation of 70-S ribosomes. The interference of IF-3 with the dynamic equilibrium of 70-S in equilibrium 50-S + 30-S occurs by binding of the factor to the 30-S subunit. The 30-S-IF-3 complex in impaired in the association reaction, which implies that IF-3 is acting as an anti-association factor. The action of IF-1 is two-fold. Firstly IF-1 increases the rate of exhcange of the ribosomal subparticles in the 70-S ribosome without changing the position of the equilibrium. Thus the spontaneous equilibrium is attained more rapidly in the presence of IF-1. This kinetic effect of IF-1 is also demonstrated in the IF-3-mediated dissociation of 70-S ribosomes. Secondly IF-1 is able to increase the IF-3-mediated dissociation. It seems likely that the explanation for the latter phenomenon must be sought in the binding of IF-1 to 70-S ribosomes, resulting in a loosening of the ribosomes structure, as well as to 30-S. IF-3 complex, thaereby slowing down the association reactions of the subunits.     

Effect of ribosomal protein L12 upon initiation factor IF-2 activities

The effect of removal of the 50S subunit proteins L7 and L12 upon initiation factor IF-2 activities is investigated. Both “coupled” and “non-coupled” GTPase activities are greatly reduced as is fMet-tRNA ribosomal binding. These activities can be restored by re-addition of L12. IF-2 activities are less affected by lack of L12 than EF-G dependent GTP hydrolysis. It is proposed that ribosomal sites for initiation factor and elongation factor -dependent GTP hydrolysis are closely associated.     

The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit

Bovine mitochondrial translational initiation factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.