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.     

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.     

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.     

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.     

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.     

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.     

The mechanism of action of initiaton factor 3 in protein synthesis,I. Studies on ribosomes crosslinked with dimethylsuberimidate

The mechanism of action of chain initiation factor 3 in translation was examined by using $\text{E. coli}$ 70S ribosomes which were covalently crosslinked with dimethylsuberimidate. Crosslinked ribosomes were inactive in AUG-dependent fMet-tRNA binding, and were not stimulated by IF-3 in poly(U) translation. IF-3 is known to be required for maximal rates of amino acid incorporation with synthetic polynucleotides at 18 mM Mg²⁺. A direct interaction of IF-3 with 70S ribosomes was demonstrated by crosslinking ¹⁴C-labeled IF-3 to 70S ribosomes. The labeled factor was also crosslinked to 30S and 50S ribosomal subunits. A model is presented proposing the mechanism of action of IF-3 on 70S ribosomes.     

The binding of dansylated initiation factor 3 to the 30-S subunit of Escherichia coli: a fluorimetric and biochemical study

Initiation factor 3 (IF-3) has been labelled using dansyl (1-dimethylaminonaphthalene-5-sulphonyl) chloride under conditions designed to preserve the biological activity of the factor. The sites of modification of the IF-3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions. However, if an IF-3 molecule bears more than one dansyl group on average then its activity is lost. The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction. Spectrofluorimetric analysis of the dansyl-IF-3 leads to the following conclusions. (a) The motion of the dansyl label does not change greatly upon binding to the 30-S subunit. (b) The label is not close enough to any tryptophan group of the ribosome in the 30-S-subunit . IF-3 complex to allow energy transfer. (c) The IF-3 chain is folded so as to bring the tyrosine groups close to the dansyl-binding sites. (d) The stoichiometry of the binding of IF-3 to 30-S ribosomal subunits is close to 1:1 and the binding constant is 2 x 10(7) M-1. IF-3 also binds non-covalently the fluorescent indicator 8-anilinonaphthalene 1-sulphonate (ANS) with an apparent binding constant of approximately 8000 M-1. An interaction between ANS and poly(A-U-G), both bound to IF-3, was demonstrated. Combining these results with those for dansyl-IF-3 leads to a model for the interaction between IF-3 and the 30-S subunit involving a combination of 'hydrophobic' and electrostatic attraction between the factor and ribosomal RNA.     

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.     

tRNA binding sites on the subunits of Escherichia coli ribosomes

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.     

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 role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

Chloroplast initiation factor 3 from Euglena gracilis. Identification and initial characterization

A chloroplast ribosome dissociation factor (IF-3chl) has been identified in whole cell extracts of Euglena gracilis. This work represents the first report of an organellar ribosome dissociation factor. E. gracilis IF-3chl facilitates the dissociation of Escherichia coli ribosomes as demonstrated by sucrose density gradient analysis. Chloroplast IF-3 stimulates initiation complex formation on E. coli ribosomes with natural mRNA from the bacteriophage MS2. In addition, IF-3chl is effective in initiation complex formation with Euglena chloroplast or E. coli ribosomes in the presence of synthetic mRNA. IF-3chl is induced 12-fold by exposure of the cells to light. The chloroplast factor has been purified 30-fold by chromatography on DEAE-cellulose and phosphocellulose. The chromatographic properties of this factor differ considerably from those of prokaryotic ribosome dissociation factors.     

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.     

Chain initiation factor 3 crosslinks to E. coli 30S and 50S ribosomal subunits and alters the UV absorbance spectrum of 70S ribosomes

We report a direct procedure to determine the proteins near the IF-3 binding site in purified 30S and 50S ribosomal subunits. This procedure introduces only limited numbers of cleavable crosslinks between IF-3 and its nearest neighbors. The cleavable crosslinking reagent, 2-iminothiolane, was used to crosslink IF-3 in place to both 30S and 50S subunits. Ribosomal proteins S9/S11, S12, L2, L5 and L17 were found, by this approach, to be in close proximity to the factor in purified IF-3-subunit complexes. In addition, IF-3 was shown to alter the ultraviolet absorbance spectrum of E. coli 70S ribosomes at 10 mM Mg2+. The magnitude of the observed difference spectrum at a constant IF-3/ribosome ratio of 1.0, is linearly dependent upon ribosome concentration over the range 5 nM - 55 nM. Titration experiments indicated that the observed effect is maximal at an IF-3/ribosome ratio of approximately 1.0. These results are taken to indicate a conformational change in the 70S ribosome induced by IF-3.     

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.     

Binding of spectinomycin by ribosomes fromEscherichia coli

The binding of the aminocyclitol antibiotic spectinomycin to 70S ribosomes and to 30S subunits fromEscherichia coli has been investigated. The association was influenced by the presence of messenger RNA. The K_d for [³H]-4 OH-spectinomycin binding to 70S ribosomes was 2×10^–7 M without mRNA (polyinosinic acid), and 1×10^–6 M with polyinosinic acid. Dissociation of the antibiotic from the ribosomes was significantly affected by the presence of a bound messenger RNA, which reduced the rate of dissociation by a factor of 5.7. The presence of mRNA did not influence the association of spectinomycin with the 30S subunit. The dissociation rate from the small subunit was comparable to the rate of dissociation from the 70S ribosome and was not affected by the presence of mRNA.     

Structure-function relationship in Escherichia coli initiation factors. Biochemical and biophysical characterization of the interaction between IF-2 and guanosine nucleotides

Equilibrium dialysis and protection from heat inactivation and proteolysis show that initiation factor 2 (IF-2) interacts not only with GTP but also with GDP and that its conformation is changed upon binding of either nucleotide. The apparent Ka (at 25 degrees C) for the IF-2 X GDP and IF-2 X GTP complexes was 8.0 X 10(4) and 7.0 X 10(3) M(-1), respectively. The lower affinity for GTP is associated with a more negative delta S0. The interaction, monitored by 1HNMR spectroscopy, is characterized by fast exchange and results in line broadening and downfield shift of the purine C-8 and ribose C-1' protons of GTP as well as of the beta, gamma-methylene protons of (beta-gamma-methylene)guanosine 5'-triphosphate. The interaction of guanosine nucleotides with IF-2 requires an H bond donor (or acceptor) group at position C-2 of the purine and involves the beta- and/or gamma-phosphate of the nucleotide while the ribose 2'-OH group or the integrity of the furan ring are less critical. IF-2 binds to ribosomal particles with decreasing affinity: 30 S greater than 70 S greater than 50 S. GTP and GDP have no effect on the binding to 70 S. GTP stimulates the binding to the 30 S and depresses somewhat the binding to the 50 S subunits; GDP has the opposite effect. These results seem to rule out that the release of IF 2 from 70 S is due to a "GDP-conformation" of the factor incompatible with its permanence on the ribosome. The rate and the extent of 30 S initiation complex formation are approximately 2-fold higher with IF-2 X GTP than with IF-2 alone. At low concentrations of IF-2 and 30 S subunits, GDP inhibits this reaction, acting as a strong competitive inhibitor of GTP (Ki = 1.25 X 10(-5)m) and preventing IF-2 from binding to the ribosomal subunit.