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Induction of L-tyrosine:2-oxoglutarate aminotransferase (EC 2,6,1,5) by N6,O²'-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) in Reuber H35 hepatoma cells reaches a maximum value between 3-5 h after addition of Bt2cAMP. We have investigated the kinetics of the increase, i.e. induction, and the decrease, i.e. the repressed state, of the tyrosine-aminotransferase-synthesizing system under these conditions. Our experimental results are as follows. 1. The repressed state of the tyrosine-aminotransferase-synthesizing system is not caused by a decrease in the intracellular cAMP concentration. 2. The repressed state is inhibited by actinomycin D(while induction is not inhibited). 3. During the repressed state no effect of dexamethasone on tyrosine aminotransferase synthesis is found, while during induction Bt2cAMP and dexamethasone act synergistically. 4. Longer starvation of the cells in serum-free medium has no influence on the kinetics of the induction/repressed state curve. From these results we have concluded that the mechanism of the transition to the repressed state of the tyrosine-aminotransferase-synthesizing system is essentially different from the mechanism of deinduction which occurs after removal of the inducer. Moreover, the repressed state of the system is a phenomenon which is induced by Bt2cAMP separately from induction at a different level of protein synthesis. [ABSTRACT FROM AUTHOR]

The adenovirus tripartite leader (TPT) 5′ untranslated region (5′UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5′UTR The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5′UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5′UTR-directed translation and poliovirus-induced host cell shut-off is discussed. [ABSTRACT FROM AUTHOR]

We wanted to discover whether the conformation of the mRNA leader sequence is involved in translational fidelity. For this purpose we constructed several mutants of Semliki Forest virus 26S mRNA and inserted AUG codons into the leader sequence. We then analyzed the results of in vitro and in vivo translation of these mRNAs, probed enzymatically the secondary structure and performed minimal energy folding of the transcripts. Our results indicate that the position of a hairpin in the leader sequence determines at which AUG codon downstream from that hairpin translation is initiated. [ABSTRACT FROM AUTHOR]

The rate of initiation of protein synthesis appears to be controlled at the level of recycling of eIF-2. In this process a new factor, designated eRF, plays an important role. The factor has been purified from the post-ribosomal supernatant and has been called formerly anti-HRI and anti-inhibitor [Amesz, H., Goumans, H., Haubrich-Morree, Th., Voorma, H. O., and Benne, R. (1979) Eur. J. Biochem. 98, 513-520]. Its effect on the initiation of protein synthesis has been studied in several assays: a small but distinct effect is found in the assay for the formation of a ternary complex between eIF-2, GTP and Met-tRNA; a 4-5-fold stimulation is obtained in assays for 40S preinitiation complex formation and in the methionyl-puromycin reaction. In the latter assay a catalytic use of eIF-2 occurs provided that eRF is present. ERF forms a complex with eIF-2 which results in a decrease of the affinity of eIF-2 for GDP, giving it the properties of a GDP/GTP exchange factor. The model stresses the catalytic use of eIF-2 in initiation provided that conditions are met for GDP/GTP exchange by a transient complex formation between eIF-2 and eRF. On the other hand, it is shown that phosphorylation of eIF-2 by the hemin-regulated inhibitor (HRI) abolishes the recycling of eIF-2, by the formation of another stable complex comprising eIF-2αP, GDP and eRF. [ABSTRACT FROM AUTHOR]

From ribosomal washes of neuroblastoma cells infected with Semliki Forest virus (SFV) a protein of Mr 33000 was purified, which comigrated with the viral capsid protein on sodium dodecyl sulfate/polyacrylamide gels and was recognized by antibodies against the capsid protein of SFV. This protein selectively inhibits the translation of host and early viral 42S mRNA in vitro, but has no effect on late viral 26S and encephalomyocarditis virus mRNA translation. Eukaryotic initiation factor 4B and cap-binding protein restore the translation of host and 42S mRNA to control levels. The capsid protein specifically prevents the binding of host mRNA into 80S initiation complexes, but has no effect on thai of late viral mRNA. We propose that the capsid protein is the component responsible for the shut-off of host protein synthesis in SFV-infected cells and for the decreased translational activity of the crude ribosomal washes from these cells. [ABSTRACT FROM AUTHOR]

Synthesis of l-tyrosine: 2-oxoglutarate aminotransferase (EC 2.6.1.5) can be induced by N6,O2'-dibutyryladenosine 3',5-monophosphate (Bt2cAMP) in Reuber H35 cell cultures. Actinomycin D fails to block this induction which indicates a target for Bt2cAMP at a posttranscriptional level. We have determined the influence of Bt2cAMP on several translational events during the tyrosine aminotransferase synthesis with the following results. (1) The number of nascent tyrosine aminotransferase chains increased, whereas no effect was measured on the number of nascent total protein chains. (2) The rate of elongation along the tyrosine aminotransferase mRNA and total mRNA is not enhanced by Bt2cAMP. (3) The induced synthesis of tyrosine aminotransferase is more sensitive to the inhibition of elongation. We conclude from our results that Bt2cAMP induces the synthesis of tyrosine aminotransferase by an increase in the rate of initiation on the tyrosine aminotransferase mRNA. [ABSTRACT FROM AUTHOR]

Binding of eukaryotic initiation factor 4C (elF-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]elF-4C was observed, provided that elF-l, elF-2, elF-3, Met-tRNAf and mRNA were included in the assay mixtures. On sucrose gradients the presence of 0.5 μM edeine was an additional prerequisite for stable binding of elF-4C. The presence of elF-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 elF-4C and the small ribosomal subunit presumably occurs at an early stage of the initiation process, since elF-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 elF-4C, when the assay was performed with purified, nearly homogeneous, initiation factors. [ABSTRACT FROM AUTHOR]

eIF-2, phosphorylated in the α subunit (Mr = 38000), was isolated from rabbit reticulocyte lysates in which protein synthesis was blocked by incubation in the presence of hemin-regulated translational inhibitor. Such eIF-2 was compared to non-phosphorylated eIF-2 from control lysates in three assay systems: (a) ternary complex formation with GTP and Met-tRNAfMet (b) methionyl-puromycin synthesis in a purified system and (c) rescue of a hemin-deficient rabbit reticulocyte lysate. No difference between the eIF-2 preparations could be detected in any of these assays. Addition of 1 pmol eIF-2 resulted in (a) the binding of 0.65 pmol [3H]Met-tRNAfMet into a ternary complex in the first assay system, (b) the synthesis of 0.6 pmol methionyl-puromycin in the second reaction and (c) the synthesis of 2.5–3.0 pmol globin in the hemin-deficient lysate, with all eIF-2 preparations tested. In the first two assay systems no phosphorylation or dephosphorylation of added eIF-2 could be detected. However, in a hemin-deficient lysate added non-phosphorylated eIF-2 was readily phosphorylated (t½ ≈ 2.5 min), whereas the phosphate group of added phosphorylated eIF-2 was rapidly exchanged (t½ ≈ 2.5 min), without significantly affecting the overall level of phosphorylation of the factor. A protein factor, antagonistic in its mode of action to the inhibitor (therefore called anti-inhibitor) was isolated from the postribosomal supernatant of rabbit reticulocyte lysate [Amesz, H., Goumans, H., Haubrich-Morree, T., Voorma, H. O. and Benne, R. (1979) Eur. J. Biochem. 98, 513–520]. This factor induced the synthesis of 20 mol globin/mol eIF-2 present in the lysate, although no reduction of the level of phosphorylation of eIF-2 was observed. On the contrary, addition of the factor to the lysate system resulted in an almost twofold increase of phosphate incorporation into eIF-2α (from 0.7 mol to about 1.2 mol phosphate/mol eIF-2). These observations support the idea that phosphorylation of eIF-2α is not the sole cause (if any) of cessation of protein synthesis in rabbit reticulocyte lysates. [ABSTRACT FROM AUTHOR]

Protein synthesis initiation factor eIF-2 was isolated and purified 4300-fold from wheat germ 23000 ⊗ g supernatant. The procedure described resulted in a 70% pure factor and involves three chromatographic steps only: (a) on Sepharose-heparin, (b) on Sepharose-concanavalin-A and (c) on phosphocellulose. The Sepharose-concanavalin-A column was required for the removal of the bulk of the proteins present in the wheat germ supernatant, its use increased the speed of the procedure considerably. The factor consists of three non-identical subunits in a 1:1:1 stoichiometric ratio with apparent Mr of 56000, 41000 and 36000, which results in an Mr of 133000 for the undissociated factor. The subunits were found to be distinct from those of the eIF-2 from reticulocytes. The factor was tested in the various model reactions of the initiation process such as ternary complex formation with Met-tRNAfMet and GTP, methionyl-puromycin synthesis and in unfractionated wheat germ 23000 ⊗ g supernatant and rabbit reticulocyte 30000 ⊗ g supernatant systems. The results support the idea of an universal mode of action of initiation factors in eukaryotic cells: i.e. the wheat germ factor stimulated the above-mentioned systems in a manner similar to eIF-2 from rabbit reticulocytes. The two factors are fully exchangeable in ternary complex formation (both factors are approximately 30% active, reach maximum levels of [³H]Met-tRNAfMet binding at 0.6 μM GTP and show very little dependence on the Mg2+ concentration) and, provided that small amounts are used, in the assay for methionyl-puromycin and in the crude rabbit reticulocyte lysate. Both factor preparations act as a substrate for a cAMP-independent protein kinase from reticulocytes (hemin-regulated translational inhibitor) which is thought to be responsible for the shut-off of protein synthesis in hemin-deprived lysates from rabbit reticulocytes: the 41000-Mr subunit of wheat germ eIF-2 and the 38000-Mr subunit of reticulocytes eIF-2 were stoichiometrically phosphorylated in vitro by this inhibitor in the presence of [γ-32p]ATP. Indeed, large amounts of the inhibitor inhibited protein synthesis in crude wheat germ extracts to some extent (25 %). However, such an inhibition could not be overcome by the addition of purified eIF-2. Furthermore, when crude wheat germ extracts were incubated with [γ-32P]ATP, the eIF-2 subunit of Mr 36000 (but not the others) became phosphorylated, although no decrease in the rate of protein synthesis could be detected. [ABSTRACT FROM AUTHOR]

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme-deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed. [ABSTRACT FROM AUTHOR]

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernatant. A comparison is made between these preparations with respect to yield and specific activity. EIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D. [ABSTRACT FROM AUTHOR]

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 fMettRNA 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. [ABSTRACT FROM AUTHOR]

The binding of labeled initiation factor IF-1 to ribosomal panicles 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 panicles 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. [ABSTRACT FROM AUTHOR]

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 or 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. [ABSTRACT FROM AUTHOR]

By means of affinity chromatography of lysates from Krebs II ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained. The fraction is eluted from the column at 350 mM KCl using a linear gradient and displays a number of activities, i. e. binding of Met-tRNAfMet to form a ternary complex with GTP; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, Met-tRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns. Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

The inhibition by thiostrepton of the initiation of protein synthesis is exerted at a different level from the inhibition of reactions mediated by EF-Tu and EF-G in the elongation of protein synthesis. The presence of thiostrepton on the 50-S subunit completely prevents the binding of the EF- Tu · GTP · aa-tRNA complex and EF-G · GTP complex to the 70-S ribosome, resulting in cessation of protein synthesis at a concentration of 1 μM thiostrepton. On the other hand, during initiation thiostrepton impairs the coupling of the 50-S subunit with the 30-S initiation complex, indirectly causing inhibition of IF-2-dependent reactions. Impairment of the coupling is strongly influenced by the conditions of incubation, Since formation of formylmethionylpuromycin and the IF-2-dependent GTP hydrolysis are inhibited to the same extent and recycling of IF-2 can take place in the presence of thiostrepton, we conclude that the basic mechanism of inhibition of initiation differs from that of inhibition of elongation. [ABSTRACT FROM AUTHOR]

In this paper the mode of action of IF-1 in 40-S initiation complex formation was studied with MS 2 RNA as messenger. Using initiation factors IF-2 and IF-3 labeled in vitro it appeared that IF-I 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-I, 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 K,, 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-I 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. [ABSTRACT FROM AUTHOR]

Ribosomal protein L 11 participates in the coupling of the 30-S initiation complex with the 50-S subunit. P37 cores, lacking L7, L8, L12, L33, L10 and L11 were reconstituted with L7 and LI0. These particles are unable to join successfully to the 30-S initiation complex, whereas reconstitution of the same cores in the presence of L7, L10 and L11 restores 60–80 % of the original coupling activity. P0 cores lacking only L7, L8, LI2 and L33 are able to carry out one round of initiation, addition of L7 resulting in complete restoration of full activity. The data obtained with these P37 core particles resemble those obtained with untreated 50-S particles carrying thiostrepton, which prevents the binding of initiation factor IF-2 into the 70-S initiation complex. It is postulated that L11 induces a niche on the ribosomal surface to facilitate the proper binding of the IF-2 GTP fMet-tRNA complex. This binding of IF-2 enables the 30-S initiation complex to join to the 50-S subunit, because of the associative ability of IF-2. If joining is impaired then both the level of fMet-tRNA binding and of the IF-2-mediated GTP hydrolysis is lowered. [ABSTRACT FROM AUTHOR]

The study undertaken in this paper on the mode of action of thiostrepton provides data which permit a more precise localization of the main target of thiostrepton. There is severe impairment of the joining of the 50-S subunit, probably carrying thiostrepton, with either the 30-S subunit or the 30-S initiation complex. The degree of impairment of this coupling is temperature dependent, being almost completely inhibited at 0 °C, whereas at 37 °C the effect is much less marked, provided that natural messenger RNA is present. The inhibition of initiation by thiostrepton is more severe in the presence of IF-1, a factor, which similar to thiostrepton, is able to shift the dynamic equilibrium of 70-S⇄50-S + 30-S more towards dissociation. By means of 14C-labeled IF-2 it is demonstrated that the binding of IF-2 into the 70-S initiation complex is prevented by thiostrepton, which seems to be the main cause for non-coupling. [ABSTRACT FROM AUTHOR]

The secondary and tertiary structure of bacteriophage RNA is possibly concerned in the regulation of the synthesis of the three proteins encoded by this RNA. The accessibility of the beginning of the three cistrons for ribosomes should therefore be different. We decided to use the aminoacyl-tRNA mug assay to obtain proof of differences in accessibility. Binding studies with aminoacyl-tRNA to ribosomes, programmed with bacteriophage MS2-RNA, revealed that, besides formylmethionyl-tRNA, alanyl-tRNA is also extremely well bound to the initiation complex. This result was taken to indicate that the beginning of the coatgene is the major ribosomal binding site with intact RNA. In experiments with other aminoacyltRNAs it was shown that seryl- and lysyl-tRNA are also bound, although to a far lesser extent as compared with the alanyl-tRNA reaction. In most experiments no significant binding of arginyl-tRNA could be recorded. Similar experiments were performed with fragmented MS2-RNA of which it is known that the secondary and tertiary structure content is much lower. Binding results with fragmented MS2-RNA revealed that the accessibility of the beginning of the different cistrons reached similar levels as indicated by the amount of binding of the following aminoacyltRNAs:alanyl-, seryl-, lysyl- and arginyl-tRNA. The synthesis of the formylmethionyl-peptides: F-Met-Ala, F-Met-Ser, F-Met-Arg, F-Met-Ala-Ser and F-Met-Ser-Lys docs occur in this binding reaction, although the efficiency of the dipeptide reaction is different for the various dipeptides involved. [ABSTRACT FROM AUTHOR]

Initiation factors recycle during the assembly of a 70-S initiation complex. At early stages the factors are present, but they are absent in the mature 70-S initiation complex. Experiments described in this paper deal with the sequence of events and the contribution of each factor in the assembly process. Initiation factors IF-1 and IF-2 bind to 30-S subunits independent of the presence of the other components involved in initiation. The fate of IF-2 during 70-S initiation complex formation has been studied in a system devoid of IF-1 and in a system in which GTP is replaced by 5′-guanylylmethylenediphosphonate (Guo-5′-P2-CH2-P). In both cases it is found that IF-2 is not released from the 70-S initiation complex. This leads to the inhibition of the elongation factor EF-Tu dependent binding of the second aminoacyl-tRNA, coded for by the messenger. The data are interpreted by assuming partly overlapping binding sites for EF-Tu and IF-2. [ABSTRACT FROM AUTHOR]

Initiation of protein synthesis in Escherichia coli is a catalytic process, provided that conditions for hydrolysis of GTP are present. This means that the initiation factors IF-1, IF-2 and IF-3 are released at a certain stage of initiation complex formation, whereafter they recycle and are reutilized in another round of initiation. Experiments presented in this paper provide evidence for the mechanism of recycling, in which the hydrolysis of GTP and IF-1 both play a predominant role. We demonstrate that binding of fMet-tRNA, which is dependent on IF-2, is a stoichiometric process in a system devoid of IF-1. Addition of IF-1 converts it into a catalytic process, however, only if GTP can be hydrolysed. No such conversion takes place with 5′-guanylylmethylenediphosphonate. The same observation is valid for the uncoupled IF-2 dependent GTPase activity. In the process of initiation IF-1 acts as a recycling promoting factor, which displays analogous properties to elongation factor EF-Ts. We postulate that a ribosomal-bound IF-2 · GDP complex is converted into an IF-2 · GTP complex, which is released and is capable of starting a new cycle of initiation. Furthermore, kinetic evidence is presented that IF-3 recycles even at the level of 30-S initiation complex formation, irrespective of whether conditions for hydrolysis of GTP are provided. [ABSTRACT FROM AUTHOR]