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 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.     

Euglena gracilis chloroplast initiation factor 2. Identification and initial characterization

The chloroplast protein synthesis factor responsible for the binding of fMet-tRNAMeti to chloroplast 30 S ribosomal subunits (IF-2chl) has been identified in whole cell extracts of Euglena gracilis. The IF-2chl activity is present in considerably higher amounts in extracts of light-grown cells than in extracts of dark-grown cells. About 90% of this activity is found in the postribosomal supernatant of the cell. Chromatography on phosphocellulose results in the partial purification of IF-2chl and separates the chloroplast factor from the cytoplasmic factor eIF-2A. The binding of fMet-tRNAMeti to chloroplast 30 S subunits is message-dependent as observed for prokaryotic systems. In addition, GTP stimulates the IF-2chl-dependent reaction 3-fold. The binding reaction shows broad monovalent and divalent cation optima. The activity of IF-2chl is stimulated 2-fold by the addition of either Escherichia coli IF-1 or IF-3, and 4-fold by the inclusion of both factors. Chloroplast IF-2 is quite active on the homologous 30 S ribosomal subunits but shows little activity on E. coli 30 S or wheat germ 40 S subunits.     

Immunological characterization of the complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Euglena gracilis chloroplast translational initiation factor 2 (IF-2chl) occurs in several complex forms ranging in molecular mass from 200 to 800 kDa. Subunits of 97 to greater than 200 kDa have been observed in these preparations. Two monoclonal antibodies were prepared against the 97-kDa subunits of IF-2chl. Both of these antibodies recognize all of the higher molecular mass forms of this factor, suggesting that these subunits are closely related. Gel filtration chromatography indicates that the higher molecular mass subunits of IF-2chl are present in the higher molecular mass complexes, whereas the smaller subunits are present in the 200-400 kDa forms of IF-2chl. Probing extracts of light-induced and dark-grown cells with the antibodies indicates that the light induction of this chloroplast factor results from the synthesis of new polypeptide rather than from the activation of an inactive precursor form of the protein. Both the higher and lower molecular mass subunits of IF-2chl are present in 30 S initiation complexes as indicated by Western analysis. The binding of IF-2chl to chloroplast 30 S ribosomal subunits requires the presence of GTP, but does not require fMet-tRNA, messenger RNA, or other initiation factors. Neither polyclonal nor monoclonal antibodies against E. gracilis IF-2chl cross-react with Escherichia coli IF-2 or with animal mitochondrial IF-2.     

Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3_chlM, molecular mass, 46402) and the portion of this factor homologous to bacterial IF-3 (IF-3_chlH, molecular mass 22829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3_chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3_chlM and IF-3_chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3_chlM and IF-3_chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3_chlH has at least 5-fold more activity than either native IF-3_chl or IF-3_chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3_chl lying outside of the homology domain may down-regulate the activity of this factor.This work was supported in part by National Institutes of Health Grant GM24963.     

Purification and characterization of the mitochondrial translocase from Euglena gracilis

The Euglena gracilis mitochondrial protein biosynthetic elongation factor G (EF-Gmt) has been purified in four steps to greater than 50% homogeneity by use of a fusidic acid affinity procedure and conventional chromatographic techniques. The purification scheme results in 1100-fold purification with about 3% recovery of the total EF-G activity present in the postribosomal supernatant prepared from whole cell extracts. E. gracilis EF-Gmt has an approximate molecular weight of 76,000, comparable to that observed for procaryotic translocases. As is the case for other translocases which have been examined, pretreatment of E. gracilis EF-Gmt with N-ethylmaleimide results in a loss of polymerization activity, indicating a role for an essential cysteine residue in catalytic activity. GDP partially protects EF-Gmt from N-ethylmaleimide inactivation. E. gracilis EF-Gmt functions well on both Escherichia coli and E. gracilis chloroplast ribosomes, but has negligible activity on wheat germ cytoplasmic ribosomes. In this respect, it differs significantly from the mitochondrial translocase of yeast which has very little activity on chloroplast ribosomes. When assayed on E. coli ribosomes, E. gracilis EF-Gmt is sensitive to the steroid antibiotic, fusidic acid, at levels similar to that required for inactivation of E. coli EF-G. It is less sensitive than E. gracilis chloroplast EF-G, and is more sensitive than Bacillus subtilis EF-G. When assayed on E. gracilis chloroplast ribosomes, the same trends in sensitivities are observed, although the exact level of fusidic acid required for inactivation is slightly altered.     

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.     

Effect of GDP on the interactions between chloroplast EF-Ts and chloroplast and E. coli EF-Tu

The effects of varying concentrations of GDP on the stability of homologous and heterologous EF-Tu:EF-Ts complexes formed with the elongation factors from the chloroplast of Euglena gracilis and from E. coli have been investigated. The complexes formed with chloroplast EF-Ts were significantly more stable to GDP-induced dissociation than those formed with E. coli EF-Ts. The complex between chloroplast EF-Tu and chloroplast EF-Ts required nearly 1,000-fold higher concentrations of GDP for dissociation than the complex between chloroplast EF-Tu and E. coli EF-Ts. The E. coli EF-Tu:chloroplast EF-Ts complex required nearly 100-fold higher levels of GDP for dissociation than the E. coli EF-Tu:E. coli EF-Ts complex.     

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.     

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.     

Euglena gracilis chloroplast elongation factor Tu. Purification and initial characterization

The chloroplast protein synthesis elongation factor Tu (EF-Tuchl) has been purified to near homogeneity from Euglena gracilis. Chromatography of the postribosomal supernatant of light-induced Euglena on DEAE-Sephadex reveals two forms of EF-Tuchl. Further purification has shown that one species consists of a complex between EF-Tuchl and a factor that stimulates its activity. The other species consists of free EF-TUchl. The factor has been purified from both chromatographic forms by taking advantage of the molecular weight shift that occurs upon disruption of the complex between EF-Tuchl and the stimulatory factor. EF-Tuchl consists of a single polypeptide chain with a molecular weight of about 50,000. EF-Tuchl is as active on Escherichia coli ribosomes as it is on its homologous ribosomes but displays no detectable activity on eukaryotic cytoplasmic ribosomes. It is stimulated in polymerization by E. coli EF-Ts and will form a complex with the prokaryotic factor that can be isolated by gel filtration chromatography. Like E. coli EF-Tu, it is sensitive to modification by N-ethylmaleimide and is inhibited by the antibiotic kirromycin. Thus, the chloroplast factor has many features that reflect the close relationship between prokaryotic and chloroplast translational systems.     

Chloroplast translational initiation factor 3. Purification and characterization of multiple forms from Euglena gracilis.

The chloroplast translational initiation factor 3 (IF-3chl) has been purified by a combination of gravity and high pressure liquid chromatographic steps. IF-3chl activity has been resolved into three forms designated alpha, beta, and gamma. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the alpha form corresponds to a single polypeptide with a molecular mass of approximately 34 kDa. The beta and gamma forms have been purified to near homogeneity, and both forms appear to function as monomers with molecular masses of about 39-42 kDa. All three forms are heat stable. All the forms of IF-3chl detected enhance the poly (A,U,G)-dependent binding of the initiator tRNA to chloroplast 30 S ribosomal subunits in the presence of Escherichia coli IF-1 and IF-2. The chloroplast factor, unlike the corresponding bacterial factor, does not have a strong RNA binding activity.     

Bovine mitochondrial ribosomes. Elongation factor specificity

The activity of bovine mitochondrial ribosomes with elongation factors from a variety of sources including the mitochondria of lower eukaryotes, chloroplasts, Gram-negative bacteria, Gram-positive bacteria, and the eukaryotic cell cytoplasm has been investigated. Bovine mitochondrial ribosomes are active with homologous mitochondrial elongation factor (EF)-G but display no activity with the mitochondrial or chloroplast translocases from the lower eukaryote Euglena gracilis, with Escherichia coli or Bacillus subtilis EF-G or with cytoplasmic EF-2. In contrast to the results obtained with the translocases, E. coli EF-Tu, B. subtilis EF-Tu, and Euglena chloroplast EF-Tu all function to a significant extent on the mitochondrial ribosomes. Cytoplasmic EF-1 has barely detectable activity on the animal mitochondrial ribosomes. The polymerization of phenylalanine by these ribosomes is dependent on poly(U), displays a rather broad Mg2+ optimum around 12 mM, and proceeds most rapidly at low monovalent ion concentrations.     

Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes

Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.     

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.     

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.     

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.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Affinity modification of Escherichia coli ribosomes with 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]-benzylidene derivative of AUGU3 in the 70S initiation complexes

Affinity labelling of the Escherichia coli ribosomes with the 2',3'-O-[4-(N-(2-chloroethyl)-N-methylamino]benzylidene derivative of AUGU3(AUGU3[14C]CHRCl) has been studied within 70S initiation complexes ribosome.AUGU3[14C]CHRCl.fMet-tRNA(Metf) and binary complex ribosome.AUGU3[14C]CHRCl. Various ways of the 70S initiation complex formation resulted in differently labelled products. Proteins S5, S7, S9, L1, L16 were thus identified as cross-linked with AUGU3[14C]CHRCl within an initiation complex obtained in the presence of initiation factors IF-1, IF-2, IF-3, whereas only proteins S5 and S7 were cross-linked within the complex obtained with the sole factor IF-2. Proteins S1, S3, L1 and L33 were labelled within the initiation complex obtained nonenzymatically but only protein S1 within the binary complex. In all complexes formed with use of initiation factors labelling of IF-2 factor was invariably observed.     

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.