Binding Of fMet-tRNA Research Articles

Studies on two initiation factors of protein synthesis from rat-liver cytoplasm.

Two initiation factors of protein synthesis designated here as IF‐S1 and IF‐S2 have been isolated from rat liver cytoplasm. The factors are separated by gel filtration of a supernatant obtained at 150000 ×g on Sephadex G‐200 from one another and are purified further by chromatography on hydroxyapatite and DEAE‐cellulose columns. Both factors stimulate poly‐(phenylalanine) synthesis at low Mg2+ concentrations in the presence of elongation factors EF‐1 and EF‐2. The factor EF‐S1 stimulates the binding of AcPhe‐tRNA and fMet‐tRNA to the small ribosomal subunit. This reaction is dependent on GTP; β,γ‐methylene‐guanosine triphosphate can replace GTP. IF‐S2 is active in the coupling of 40‐S and 60‐S subunit to active 80‐S initiation complexes. This activity depends on poly(U), on Phe‐tRNA and partially on GTP. Similarities and possible analogies between the initiation factors described here with those described by other authors arc discussed.

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

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

Ribosomal protein S21 is required for full activity in the initiation of protein synthesis.

The role of the 30 S ribosomal protein S 21 in ribosome function, specifically in relation to the initiation of polypeptide synthesis, was studied. Reconstituted particles using 16 S RNA and all purified 30 S proteins, but omitting S21. The reconstituted particles ([-S 21] particles) sedimented at 30 S and contained all the 30 S proteins except S 21 in approximately the same concentration as control reconstituted particles ([Σ Si] particles). In several functional assays including AUG-directed fMet-tRNA binding, [-S 21] particles were only 33 to 44% as active as complete particles. The addition of pure S 21 to [-S 21] particles showed 2 to 3 fold stimulation in AUG-directed fMet-tRNA binding using purified initiation factors IF 1 and IF2, assayed either in the presence or absence of 50 S subunits. Maximum stimulation was observed when approximately 0.5 to 1.0 mole of S 21 per mole of [-S 21] particles were added. We conclude that, contrary to the report of van Duin et al. (1972), S 21 does not inhibit AUG-directed fMet-tRNA binding, and that S 21 is required for full activity of the ribosomes in the initiation of polypeptide synthesis. Thus, the results published by van Duin et al. cannot be taken to support the postulated heterogeneity of the ribosome population in vivo. Possible reasons for the discrepancy between our conclusion and that of van Duin et al. are discussed.

Interference of virginiamycin M with the initiation and the elongation of peptide chains in cell-free systems

1. The action of virginiamycin M on the initiation and elongation steps of polypeptide synthesis in the cell-free systems of Escherichia coli has been analysed using washed ribosomal particles, phage RNA as a natural messenger and highly purified initiation and elongation factors. 2. In systems containing optimal concentrations of the initiation factor IF-1, IF-2 and IF-3 none of the reactions leading to the formation of the mRNA · 70-S · fMet-tRNA initiation complex were found to be affected by the antibiotic. Gross interference with the IF-3-dependent dissociation of 70-S ribosomes and with the recycling of IF-3 was not detected either. 3. Pronounced inhibition of fMet-tRNA binding to 70-S ribosomes by the antibiotic occurs at low concentrations of IF-2. 4. The main actions exerted by virginiamycin M at optimal concentrations of the initiation factors are the inhibitions of the following reactions: [MS2-RNA·70-S·fMet-tRNA] + [EF-Tu·alanyl-tRNA·GTP] → [MS2-RNA·70-S·fMet-tRNA·alanyl-tRNA] + EF-Tu + GDP + P i [MS2-RNA·70-S·fMet-tRNA] + puromycin→fMet-puromycin + [MS2-RNA·70-S·tRNA] 5. Virginiamycin M mediates the release of substantial amounts of fMet-alanyl-tRNA subsequent to the interaction of alanyl-tRNA with the 70-S initiation complex (without addition of EF-G). It is assumed that the detachment occurred from the A site.

Function of individual 30S subunit proteins of Escherichia coli. Effect of specific immunoglobulin fragments (Fab) on activities of ribosomal decoding sites.

Specific anti-30S protein immunoglobulin G fragments (Fab) were used to determine the contribution of each of the 30S ribosomal proteins to: (1) polyphenylalanine synthesis, (2) initiation factor-dependent binding of fMet-tRNA, (3) T-factor-dependent binding of phenylalanyl-tRNA, and (4) fixation of radioactive dihydrostreptomycin. Twenty of the 21 possible antibodies (antibody against S17 excepted) were used. In conditions where all the 30S proteins were accessible to Fabs, all of these monovalent antibodies strongly inhibited polyphenylalanine synthesis in vitro. Antibodies against S4, S6, S7, S12, S15, and S16, however, showed a weaker effect.30S proteins can be classified into four categories by their contributions to the function of sites "A" and "P": class I appears nonessential for tRNA positioning at either site (S4, S7, S15, and S16); class II includes proteins whose role in initiation is critical (S2, S5, S6, S12, and S13); class III (S8, S9, S11, and S18) corresponds to proteins whose blockade prevents internal (elongation factor Tudependent) positioning; and class IV includes entities that are essential for activities of both "A" and "P" sites (S1, S3, S10, S14, S19, S20, and S21). Dihydrostreptomycin fixation to the 30S or 70S ribosomes was inhibited by antibodies against S1, S10, S11, S18, S19, S20, and S21, but only weakly by the anti-S12 (Str A protein) Fab. The significance of these results is discussed in relation to 30S protein function, heterogeneity, and topography.

2] Electron microscopic studies of chain initiation complexes and polysomes

Publisher Summary Formation of the chain initiation complex (mRNA · ribosome · fMettRNA) that purified E. coli ribosomes and dependence of this reaction on the chain initiation factors was studied by centrifugation in sucrose density gradients and by electron microscopy. The three chain initiation factors are required for maximum binding of natural mRNA and fMettRNA to the 70 S region. A complex involving mRNA and fMet-tRNA may also be formed on the 30 S ribosomal subunits. Both sucrose density gradient analyses and electron microscopic observations indicate the presence of one primary initiation site on intact phage RNA.

8] Purification and properties of interference factor i: a cistron-specific translation control factor and a subunit of phage RNA replicase

Interference factors are characterized by their mRNA-specific effects on IF3 activity, in initiating the translation of various mRNA's. The assay purification and properties of interference factor i 2.3 (i-a) are described in this chapter. The assay is based on the fact that this protein inhibits translation of native MS2 RNA, but not T4 mRNA. The inhibition of MS2 RNA translation is because of the fact that factor i blocks ribosome binding and initiator fMet-tRNA binding at the coat protein initiation site, which is the only site exposed on native MS2 RNA. When the MS2 RNA is first unfolded by mild formaldehyde treatment, initiation can take place at the three cistrons independently, and because factor i blocks only coat protein synthesis, this template is actively translated in the presence of factor i. The assay of factor i is carried out by measuring the differential effect on at least two different mRNA templates.

Role of Guanine Nucleotides in Protein Synthesis. Elongation Factor G and Guanosine 5′-Triphosphate,3′-Diphosphate

The possible role of guanosine 5'-triphosphate,3'-diphosphate (pppGpp) in protein synthesis by Escherichia coli ribosomes and protein factors was examined. Although pppGpp could effectively substitute for GTP in reactions catalyzed by initiation factor 2 (ribosomal binding of fMet-tRNA and formation of N-formylmethionylpuromycin) and elongation factor T (ribosomal binding of Phe-tRNA and formation of dipeptidyl-tRNA), pppGpp poorly supported polyphenylalanine synthesis. The interaction of elongation factor G with pppGpp was, therefore, examined in detail. The nucleotide was found to be almost without activity in the translocation reaction, as measured by formation of N-acetylphenylalanyl-phenylalanylpuromycin. Nevertheless, the rate of the catalytic hydrolysis of pppGpp to guanosine 5'-diphosphate,3'-diphosphate by elongation factor G and ribosomes was about 30% of the rate of hydrolysis of GTP, a rate of hydrolysis that significantly exceeded the rate of translocation with GTP. Moreover, the rates of the fusidic acid-dependent, elongation factor G-dependent binding of pppGpp and ppGpp to ribosomes were about 75 to 85% the rates of GTP and GDP binding, respectively. We also found that dGTP could substitute for GTP in all reactions examined.

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg 2+ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide. Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin. The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.

Reconstitution of Escherichia coli 30 S Ribosomal Subunits from Purified Molecular Components

Abstract Reconstitution of 30 S ribosomal subunits from 16 S RNA and a mixture of purified individual 30 S ribosomal proteins has been studied. Proteins from the 30 S ribosomal subunit of Escherichia coli were purified by a combination of phosphocellulose and DEAE-chromatography, and Sephadex gel filtration. The proteins purified correspond to the 21 proteins generally accepted as 30 S proteins, with the exception of two proteins, P3b and P3c, which correspond to the protein S6 studied by other workers. P3b and P3c are closely related, and one is probably a derivative of the other. Using a mixture of these purified proteins, reconstitution of functionally active 30 S subunits has been demonstrated. Reconstituted particles had higher activities in poly(U)-directed polyphenylalanine synthesis than reference 30 S particles in several experiments. The functional activity of reconstituted particles was also examined in several other assays; these included natural messenger RNA-directed polypeptide synthesis, poly(U)-directed Phe-tRNA binding, AUG-directed fMet-puromycin formation, AUG-directed fMet-tRNA binding, and the binding of termination codon UAA in the presence of chain termination factors. In all cases, activities comparable to reference 30 S subunits were observed. The sedimentation properties and the protein composition of reconstituted particles were also similar to 30 S ribosomal subunits. The kinetics of reconstitution using purified protein mixtures was essentially identical with those of reconstitution using unfractionated 30 S proteins. These results strongly suggest that 21 purified 30 S proteins together with 16 S RNA are sufficient to reconstitute 30 S subunits, and that no essential 30 S components were lost during the fractionation and purification of the 30 S proteins. Single component omission experiments indicated that all purified proteins, except P1(S1) and P3b,c(S6), are required for full functional activity. A requirement for P9a(S16) has been shown in some, but not all, experiments. P3b,c(S6) and P9a(S16) have been shown to be involved in the reconstitution reaction in other experiments (Mizushima, S., and Nomura, M. (1970) Nature 226, 1214; Nomura, M. (1973) Science 179, 864) and therefore are 30 S components. It is still not clear whether P1(S1) should be considered a true 30 S protein or a ribosomal-associated factor.

Non-enzymic binding of formylmethionyl-transfer RNA f to Artemia satina ribosomes

40 S ribosomal subunits of Artemia salina embryos can bind formylmethionyl-transfer RNA f non-enzymically, i.e., in the absence of initiation factors. This, like the enzymic reaction, is largely AUG-dependent. Much more fMet-tRNA f is bound by 80 S ribosomes but, in this case, a large fraction (about two-thirds) of the binding is AUG-independent. Whereas the AUG-dependent binding is very sensitive to edeine, a potent initiation inhibitor, the AUG-independent binding is resistant to this antibiotic. Virtually all of the bound fMet-tRNA f is in all cases capable of reacting with puromycin to form fMet-puromycin; hence the bound aminoaoyl-tRNA is in the peptidyl (donor) site of the 80 S ribosome. Non-acylated tRNAs also bind to this site with high affinity in a codon-independent reaction and block the 80 S binding of fMet-tRNA f. The properties of the peptidyl site are consistent with a non-decoding site which harbors the initiator aminoacyl-tRNA, when the 80 S initiation complex is formed, and to which every molecule of tRNA remains temporarily attached following peptide bond synthesis.

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 f 2 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.

Chain Initiation Factor 2: PURIFICATION AND PROPERTIES OF TWO SPECIES FROM ESCHERICHIA COLI MRE 600

Abstract Chain initiation factor 2 was resolved into two proteins, designated IF-2-α and IF-2-β, by ion exchange chromatography in 6 m urea. The molecular weight of IF-2-α was 98,000 and that of IF-2-β was 83,000. In the presence of IF-1 and IF-3, both IF-2-α and IF-2-β promoted the binding of fMet-tRNA to ribosomes with either ApUpG, GpUpG, or R17 RNA as messenger. The activities of IF-2-α and IF-2-β were additive. Neither protein was protected by GTP against heat inactivation, nor could a stable complex of GTP, IF-2 and fMet-tRNAf be isolated by Sephadex G-50 chromatography. The binding of pure fMet-tRNAf to ribosomes was stimulated by both species of IF-2.

Recycling of initiation factors IF-1, IF-2 and IF-3.

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.

Polypeptide chain initiation in eukaryotes: IV. Purification and properties of supernatant initiation factor from Artemia salina embryos

The eukaryotic supernatant initiation factor, described in earlier publications from this laboratory, has been isolated and purified over 3000-fold, to about 70 to 80% purity, from extracts of embryos of the brine shrimp Artemia salina. The native protein appears to consist of two equal subunits, each weighing approximately 74,000 daltons. Like the bacterial initiation factor IF2, its prokaryotic counterpart, the Artemia factor promotes the AUG-dependent binding of fMet-tRNA, or the poly (U)-dependent binding of N-acetyl-Phe-tRNA, to the small ribosomal subunit. However, unlike IF2, the reaction is GTP-independent and the factor functions catalytically for one molecule may promote the binding of up to 12 molecules of fMet-tRNA to 40 s subunits at 0 °C.

F2 RNA structure and peptide chain initiation: fMet-tRNA binding directed by methoxyamine-modified unfolded or native-like f2 RNAs

f2 RNA in which all exposed, unpaired cytosines are modified with methoxyamine retains an unchanged ordered structure. This RNA shows the same capacity to direct fMet-tRNA binding to E. coli ribosomes as unmodified f2 RNA. As this binding corresponds mainly to the coat protein initiation site, our results suggest that unpaired cytosines present in the coat protein initiation fragment are not involved in the specific recognition of that fragment by ribosomes. When f2 RNA is treated with methoxyamine in the presence of guanidine-HCl the resulting modified f2 RNA becomes irreversibly unfolded. Such RNA stimulates fMet-tRNA binding about 50 times more efficiently than native f2 RNA. E. coli ribosomal RNA when unfolded under the same conditions also stimulates very efficiently the binding of fMet-tRNA.

Vernamycin A inhibits the non-enzymatic binding of fMet-tRNA to ribosomes

The ribosomal site of action of the antibiotic vernamycin A was studied by investigating the effect of the drug on the non-enzymatic binding of fMet-tRNA. 1. 1. Vernamycin A inhibits the non-enzymatic binding of fMet-tRNA to 70-S ribosomes and to 50-S subunits, and can displace previously bound fMet-tRNA from 70-S ribosomes. 2. 2. Vernamycin A inhibits the binding of fMet-tRNA to a puromycin reactive site (P site), which is not affected by tetracycline. 3. 3. Although vernamycin A inhibits the puromycin reaction in model systems it does not inhibit peptide bond formation occurring on polysomes. Consequently, the antibiotic probably inhibits peptide bond formation in model in vitro systems not by directly inhibiting peptide bond formation, but by inhibiting the binding of the tRNA to the ribosome. 4. 4. The data indicate that the ribosomal binding sites for vernamycin A on the one hand and carbomycin B, chloramphenicol, erythromycin, leucomycin A 3, spiramycin III and tylosin on the other hand, are different.

Aminoacyl-transfer RNA binding to active 30 s subunits: Effect of 50 s subunits and a new role for elongation factor T

It has been formerly shown that low concentrations of Mg 2+, as are used to dissociate ribosomes into subunits, render the ribosomes inactive in binding of aminoacyl-transfer RNA. Activity is restored on heating the ribosomes under appropriate salt conditions. We have now studied the binding of aminoacyl-tRNA to fully activated ribosomes thus eliminating any possible activation during the binding reaction. The requirements and kinetics of the reaction as revealed are therefore those of the binding reaction and not of the activation step. The major findings were as follows. 1. (i) Non-enzymic binding of Phe-tRNA to 30 s subunits proceeds efficiently in the cold and at Mg 2+ concentrations as low as 5 mM. 2. (ii) The instability of the 30 s messenger RNA-aminoacyl-tRNA complex is suggested to be due to transformation of the 30 s subunits into a form incapable of further binding. This inactivation occurs during the binding reaction and depends on it. 50 s subunits prevent the inactivation and restore binding ability to seemingly inactive 30 s subunits. The observed stimulation of aminoacyl-tRNA binding to 30 s subunits by 50 s subunits may thus be due to stabilization of the active form of the ribosome by 50 s subunits and not necessarily to the formation of an additional binding site. 3. (iii) 50 s subunits inhibit the initial rate of binding of different aminoacyl-tRNA's, indicating that such inhibition is not a unique property of fMet-tRNA binding. 4. (iv) T factor stimulation of aminoacyl-tRNA binding is related to the inhibition of the reaction by 50 S subunits. The results indicate that binding to isolated 30 s subunits represents under the given conditions the full binding potential of the ribosomes. 50 s subunits suppress this binding and the presence of T factor restores it to the level seen with 30 s subunits alone. Thus, the Tu-GTP-aminoacyl-tRNA complex appears to overcome the inhibition exerted by 50 s subunits.

Functional heterogeneity of the 30S ribosomal subunit of Escherichia coli. II. Effect of S21 on initiation.

Native 30S ribosomal subunits fromEscherichia coli are deficient in fractional protein S21, which is present on the monosome and polysome-derived 30S subunits. The presence of S21 prevents the binding of Fmet-tRNA if and only if 50S subunits are present. In contrast, proteins S2, S3 and S14 stimulate the binding of Fmet-tRNA. These results have been used to rationalize other data concerning the mechanism of Fmet-tRNA binding by ribosomes. In addition, the present data indicate that the 30S ribosomal subunits are heterogeneousin vivo as well asin vitro.

Characterization of the Ribosome-dependent Guanosine Triphosphatase Activity of Polypeptide Chain Initiation Factor IF 2

Abstract 1. Polypeptide chain initiation factor IF 2 exhibits a potent ribosome-dependent GTPase activity. This activity has been characterized with regard to various biochemical parameters. The reaction requires the presence of both 30 S and 50 S ribosomal subunits. The stoichiometry of the reaction is GTP → GDP + Pi. The reaction requires Mg++ and is highly stimulated by NH4+ or K+. Besides GTP, dGTP is also active in the reaction while other nucleoside triphosphates are inactive. K8 for GTP is 7.7 x 10-6 m; GDP is a potent competitive inhibitor with a Ki of 2.0 x 10-6 m. No exchange between GTP and either GDP or Pi can be detected. 2. The GTPase activity of IF 2 is distinct from that of either of the peptide chain elongation factors, T and G. 3. Evidence is presented that the GTPase and fMet-tRNA binding activities exhibited by IF 2 are mediated by the same protein; the GTPase and fMet-tRNA binding activities of IF 2 cochromatograph on phosphocellulose and DEAE-Sephadex and cosediment on glycerol gradient centrifugation. Furthermore, both activities display similar sensitivities to p-hydroxymercuribenzoate, to heat, and to inhibition by GDP. 4. Under conditions of fMet-tRNA binding to ribosomes to form the 70 S initiation complex, stimulation of IF 2-dependent GTP hydrolysis (coupling) can be demonstrated.