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.     

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes was obtained using a functional analogue of aminoacyl-tRNA. Since elongation factor Tu (EF-Tu) screens both the modified aminoacyl-tRNAs and the ribosomal complexes for active particles, only functional macromolecular complexes are examined. This approach also provides an unequivocal identification of the transfer RNA binding site from which affinity labeling occurs. N^ε-bromoacetyl-Lys-tRNA was prepared by covalently attaching an electrophilic group to the side-chain of the amino acid. This chemical modification did not interfere with function, since the ?BrAcLys-tRNA participated successfully in EF-Tu and poly(rA)-dependent binding to ribosomes, peptide bond formation, and elongation factor G (EF-G)-mediated translocation. Affinity labeling of ribosomal RNA was observed only in those incubations which contained both EF-Tu and EF-G. The crosslinking of ?BrAcLys-tRNA to 23 S rRNA was found even if fusidic acid was added to the incubation before EF-G. The dependence of the covalent reaction on EF-G demonstrates, unambiguously, that a reactive residue of 23 S rRNA is located adjacent to the 3′ end of the functionally defined P site. Similarly, the affinity labeling of proteins L13/14/15, L2, L32/33, and L24 required EF-G-dependent translocation of ?BrAcLys-tRNA into the P site. Protein L27 was alkylated following the EF-Tu-dependent binding of ?BrAcLys-tRNA to the ribosome, and the extent of affinity labeling was stimulated by the addition of EF-G to the incubation. Double-label dipeptide experiments confirmed that affinity labeling occurred from functional tRNA binding sites by demonstrating that the same ?BrAcLys-tRNA which reacted covalently with 23 S rRNA or a ribosomal protein could also participate in peptide bond formation. Finally, the ribosome affinity labeling obtained with ?BrAcLys-tRNA · EF-Tu · guanylylimidodiphosphate differed little from that obtained with ?BrAcLys-tRNA · EF-Tu · GTP. This work constitutes the first direct examination of the aminoacyl ends of the EF-Tu-dependent conformational states of the ribosomal complex, and demonstrates the potential value of functional Lys-tRNA analogues with different probes attached to the lysine side-chain.     

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

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

Initiation 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.     

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

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

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

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

Ribosomal protein L1 from Escherichia coli. Its role in the binding of tRNA to the ribosome and in elongation factor g-dependent gtp hydrolysis

Two Escherichia coli mutants lacking ribosomal protein L1, previously shown to display 40 to 60% reduced capacity for in vitro protein synthesis (Subramanian, A. R., and Dabbs, E. R. (1980) Eur. J. Biochem. 112, 425-430), have been used to study partial reactions of protein biosynthesis. Both the binding of N-acetyl-Phe-tRNA to ribosomes and the 6 to 8-fold stimulation of the elongation factor G (EF-G)-dependent GTPase reaction by mRNA plus tRNA, assayed in the presence of wild type 30 S subunits, were low with L1-deficient 50 S subunits. Addition of pure protein L1 to the assay restored both reactions to 100% of the control. By contrast, the basic EF-G GTPase reaction in the absence of mRNA and tRNA was not at all affected (mRNA alone had no effect). None of the following partial reactions were more than moderately modified by the lack of protein L1: binding to ribosomes of EF-G.GDP plus fusidic acid; the translocation reaction catalyzed by EF-G plus GTP; poly(U)-dependent binding to ribosomes of Phe-tRNAPhe (whether dependent on elongation factor Tu plus GTP or not); and the EF-Tu-dependent GTPase activity. It is concluded that protein L1 is involved in the interaction between ribosomes and peptidyl-tRNA (or tRNA) in the peptidyl site and consequently in the ribosomal GTPase activity depending on the simultaneous action of tRNA and EF-G.     

Preparation and properties of a Met-tRNAf binding factor from rat liver and rat hepatoma.

A Met-tRNAf binding factor (IF-2) from the microsomal fraction of rat liver and rat hepatoma ascites cells was partially purified by ammonium sulphate fractionation, DEAE-cellulose and phosphocellulose chromatography. The factor binds [3H]Met-tRNAf only in the presence of either GTP or GMPPCP. Maximal binding takes place at 37 degrees C and in the absence of Mg++. The factor is specific for Met-tRNAf and does not bind Phe-tRNA from rat liver or from E. coli. The ternary complex [Met-tRNAf . IF-2 . GTP1 binds to 40 S ribosomal subunits from rat liver in the absence of mRNA or poly(A, G, U) without GTP hydrolysis. GDP as well as aurintricarboxylic acid inhibit the ternary complex formation. Both factors are rapidly inactivated by N-ethylmaleimide treatment and by preincubation at 45 degrees C. Heat inactivation is partially prevented by GTP and GDP. With regard to the functional properties there are no significant differences between IF-2 from normal liver and hepatoma cells. On the other hand heat denaturation compared to the rat liver factor, which may be due to differences in contaminating proteins.     

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.     

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.     

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.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase 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 f₂ RNA. The formation of the 70S initiation complex is dependent upon IF-2 and is stimulated by IF-1. The requirement for IF-3 is not alleviated by high concentrations of the synthetic templates.     

Interaction of elongation factor EF-Tu with γ-amides of GTP and β-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate

New types of azidoaryl analogs of GTP: γ-(4-azido)anilide of GTP (I), γ-(N-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: β-(4-azido)anilide of GDP (III), β-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N′-β-(4-methylmorpholinium)- ethylcarbodiimidep-toluene sulfonate and the respective amine. The analog of GTP bearing at the γ-phosphate an alkylating 2-chloroethylamino group: γ-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A. and Samukov, V.V. (1976) FEBS Lett. 70, 105–108). Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu·GDP with the respective K_i values 3.9·10^?7 M (I), 2.9·10^?8 M (II), 6.9·10^?7 M (III), 5.0·10^?7 M (IV) and 3.8·10^?8 M (V) relative to GDP. constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5·10^?6 M, 3.4·10^?7 M and 4.6·10^?8 M, respectively, or approx. 1740-, 70- and 9-times greater than that of GDP. GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex.     

Purification and properties of a methionine formylmethionyl-tRNA-binding initiation factor from pig liver

(i) A factor, EIF-2, that binds methionyl-tRNA_f^Met in the presence of GTP has been isolated from pig liver. (ii) Dodecylsulfate-gel electrophoresis and sedimentation equilibrium centrifugation indicate that the factor has a molecular weight of 122,000 and that it consists of three unequal subunits. (iii) The apparent K_D for binding of methionyl-tRNA_f^Met varies with factor concentration. GTP participates in the binding with a K_D of 0.5 μm. β,γ-Methylene-guanosine triphosphate supports 40% of the binding observed with GTP. GDP is a competitive inhibitor with a K_i of 0.2 μm. The optimal, free Mg²⁺ concentration is approximately 50 μm. GTP and Mg²⁺ stabilize the factor against thermal inactivation and inactivation by N-ethyl maleimide. (iv) The factor is required for the formation of a sucrose gradient-stable complex between methionyl-tRNA_f^Met and the 40S ribosomal subunit. The presence of template is not necessary, but poly(A,U,G) increases the binding observed 1.5-fold. (v) The factor markedly stimulates synthesis in a reconstituted protein-synthesizing system with globin messenger RNA as template.     

Significance of accumulation of the alarmone (p)ppGpp in chloroplasts for controlling photosynthesis and metabolite balance during nitrogen starvation in Arabidopsis

Photosynthesis Research - The regulatory nucleotides, guanosine 5′-triphosphate 3′-diphosphate (pppGpp) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp), were originally...     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

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.