Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.     

Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes.

Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.     

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.     

Threonyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli: Subunit Structure and Genetic Analysis of the Structural Gene by Means of a Mutated Enzyme and of a Specialized Transducing Lambda Bacteriophage

Threonyl-transfer ribonucleic acid synthetase (ThrRS) has been purified from a strain of Escherichia coli that shows a ninefold overproduction of this enzyme. Determination of the molecular weight of the purified, native enzyme by gel chromatography and by polyacrylamide gel electrophoresis at different gel concentrations yielded apparent molecular weight values of 150,000 and 161,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band of 76,000-dalton size. From these results an alpha(2) subunit structure can be inferred. A mutant with a structurally altered ThrRS, which had been obtained by selection for resistance against the antibiotic borrelidin, was used to map the position of the ThrRS structural gene (thrS) by P1 transductions. It was found that thrS is located in the immediate neighborhood of pheS and pheT, which are the structural genes for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase, the gene order being aroD-pheT-pheS-thrS. A lambda phage that was previously shown to specifically transduce pheS, pheT, and also the structural gene for the translation initiation factor IF3 can complement the defect of the altered ThrRS of the borrelidin-resistant strain. This phage also stimulates the synthesis of the 76,000, molecular-weight polypeptide of ThrRS in ultraviolet light-irradiated. E. coli cells. These results indicate that the genes for ThrRS, alpha and beta subunits of phenylalanyl-tRNA synthetase, and initiation factor IF3 are immediately adjacent on the E. coli chromosome.     

Production and epitope characterization of mAbs specific for translation factor IF1

Initiation of protein synthesis in bacteria relies on the presence of three translation initiation factors, of which translation initiation factor IF1 is the smallest having a molecular weight of only 8.2 kDa. In addition to its function in this highly dynamic process, the essential IF1 protein also functions as an RNA chaperone. Despite extensive research, the exact function of IF1 in translation initiation has not yet been determined, and the research in the function of the factor has in some areas been impeded by the lack of monoclonal antibodies specific for this protein. Several attempts to induce immune response in mice with wild-type IF1 for the production of antibodies have failed. We have now succeeded in producing monoclonal antibodies specific for IF1 by applying a new immunization strategy involving an antigen combination of IF1 coupled to glutathione S-transferase (GST) and a recombinant dimer of IF1. This resulted in the generation of 6 IgG, 2 IgM, and 1 IgA anti-IF1 antibodies, which can be used in ELISA screening and Western immunoblots. We also provide a mapping of the functional epitopes of the generated anti-IF1 monoclonal antibodies by screening the antibodies for binding to IF1 proteins mutated at single amino acid positions.     

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.     

Superexpression and fast purification of E coli initiation factor IF2.

For the production of large quantities of E coli initiation factor IF2 we have constructed an improved overexpression system. The gene infB was cloned into the thermo-inducible runaway plasmid pCP40 [1] and subsequently transformed into the E coli strain C600[pcI857]. In this system the expression of infB is under the control of the strong promoter lambda PL and the cells carry the plasmid pcI857, which contains a thermosensible lambda cI repressor. Overexpression of IF2, which is approximately 30 times higher than the expression in wild-type-cells, is induced at 42 degrees C and continues for 2 h at 37 degrees C. From these cells pure and active IF2 was obtained using a novel 3-step FPLC-procedure consisting of ion-exchange liquid chromatography on Q-sepharose HP, MonoQ and MonoS. In approximately 8 h, 5 mg of pure and active IF2 can be obtained from 10 g overproducing cells. This corresponds to 5 mg of IF2 per litre of medium. The purification was monitored by Western immunoblotting and the activity of the purified factor was tested by measuring the stimulation of binding of the initiator fMet-tRNA(Met)f to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger RNA. Compared with previous methods our purification procedure avoids the use of materials such as DEAE-cellulose and phosphocellulose which have relatively poor flow rates. In addition to the higher flow capacity of Q-sepharose HP, this new matrix can be loaded with an S30 supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein synthesis initiation factor 2 from chicken reticulocytes: purification by affinity chromatography and preliminary characterization

Affinity chromatography on beta,gamma-methylene guanosine 5'-triphosphate-Sepharose was used to purify protein synthesis initiation factor eIF-2 from chicken reticulocytes. Gel filtration of the purified factor gave a molecular weight of 150,000, whereas electrophoresis of the purified factor on polyacrylamide gel containing sodium dodecyl sulfate revealed three non-identical subunits with apparent molecular weight of 57,000, 41,000 and 33,000. With Met-tRNA and GTP, the factor formed a ternary complex which would bind the 40S ribosomal subunits. Treatment of the factor with N-ethylmaleimide resulted in a loss of activity. Two sulfhydryl groups per eIF-2 molecule were essential for activity.     

Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms.

Two forms of E. coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2 beta. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.     

Immunochemical analysis of molecular forms of protein synthesis initiation factors in crude cell lysates of Escherichia coli

Three initiation factors (IF1, IF2, and IF3) have been highly purified from Escherichia coli and extensively characterized, but little is known about the molecular forms of these proteins as they occur in vivo. We have analyzed molecular-weight and charge forms in crude cell lysates by polyacrylamide gel electrophoresis followed by immunoblotting with antibodies specific for the initiation factors. Freshly grown bacterial cells were lysed by sonication in buffer containing sodium dodecyl sulfate, and the lysate was fractionated by gel electrophoresis. Proteins from the gel were electrotransferred to a nitrocellulose sheet which was treated with a specific rabbit antiserum followed by radiolabeled Staphylococcus aureus protein A. Autoradiography showed only one major band each for IF1 and IF3, exactly corresponding to the isolated factors. For IF2, two molecular-weight forms were detected which were identical with purified IF2a and IF2b. No evidence for precursor forms was found. Two-dimensional gel analysis showed no charge heterogeneity for IF1, IF2a, and IF3, but multiple forms were seen for IF2b. Analysis of phosphoproteins from cells grown in radioactive phosphate medium ruled out the possibility that phosphorylation occurs on the initiation factors, elongation factors, or ribosomal proteins.     

The N-terminal domain (IF2N) of bacterial translation initiation factor IF2 is connected to the conserved C-terminal domains by a flexible linker

Bacterial translation initiation factor IF2 is a multidomain protein that is an essential component of a system for ensuring that protein synthesis begins at the correct codon within a messenger RNA. Full-length IF2 from Escherichia coli and seven fragments of the protein were expressed, purified, and characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) methods. Interestingly, resonances of the 6 kD IF2N domain located at the extreme N terminus of IF2 can be clearly identified within the NMR spectra of the full-length 97-kD protein. (15)N NMR relaxation rate data indicate that (1) the IF2N domain is internally well ordered and tumbles in solution in a manner that is independent of the other domains of the IF2 protein, and (2) the IF2N domain is connected to the C-terminal regions of IF2 by a flexible linker. Chemical shifts of resonances within the isolated IF2N domain do not significantly differ from those of the corresponding residues within the context of the full-length 97-kD protein, indicating that IF2N is a structurally independent unit that does not strongly interact with other regions of IF2. CD and NMR data together provide evidence that Domains I-III of IF2 have unstructured and flexible regions as well as substantial helical content; CD data indicate that the helical content of these regions decreases significantly at temperatures above 35 degrees C. The features of structurally well-ordered N- and C-terminal domains connected by a flexible linker with significant helical content are reminiscent of another translation initiation factor, IF3.     

A high molecular weight platelet aggregating factor in Crocus sativus

Bulbs of Crocus sativus, variety Cartwrightianus contain a protein factor with aggregating properties on human platelets. This factor was purified by different chromatographic techniques and shows a molecular weight of 42 000, as it was estimated by Sephadex G-75 column chromatography and sodium dodecyl sulfate (SDS) polyacrylamide slab gel electrophoresis.     

Actin co-purifies with RNA polymerase II.

RNA polymerase II, [EC2.7.7.6], from the slime mold Physarum polycephalum, purified over 4000-fold can contain a protein with an apparent molecular weight of 46,000. This protein is separated from the putative subunits of RNA polymerase II by polyacrylamide gel electrophoresis under non-denaturing conditions, and by chromatography on phosphocellulose. In this report we identify the protein as actin, and we point out that polypeptides of this apparent molecular weight which have been found associated with RNA polymerase II purified from other sources may also be actin from these organisms.     

Purification and properties of a protein from the nuclear fraction of HeLa cells binding to a cloned fragment of human satellite DNA III

In nuclear extract of HeLa cells two proteins were identified having the specific binding activity to cloned 1.8kb fragment of human satellite DNA III (HS3). One of the satellite binding proteins (SBP1) purified by column chromatography using DEAE-, phospho- and DNA-cellulose steps interacted also with adenovirus 5 replication enhancer (ARE), another protein (SBP2) was separated during phosphocellulose chromatography from ARE-binding protein. It is suggested that SBP1 is possibly identical to the nuclear factor I purified earlier from the nuclear extract of HeLa cells by other authors.     

Purification of a novel eIF-2 alpha protein kinase from calf brain

A new eukaryotic initiation factor 2 kinase has been purified for the first time from calf brain cytosol. The purification of a nonabundant novel protein kinase activity, designated as PKI, that phosphorylates the alpha subunit of eukaryotic initiation factor 2 is described. The protein kinase activity was assayed using purified initiation factor 2 as a substrate and was purified by ammonium sulphate precipitation, conventional chromatography in heparin-Sepharose and phosphocellulose and by high performance size exclusion and anion exchange chromatographies. The protein kinase activity elutes in the region of 140,000 in the size exclusion chromatography and is associated with two different polypeptides a and b, with relative molecular masses of 38,000 and 20,000 and an approximate ratio of 2.5-3.0:1. The protein kinase does not phosphorylate casein or histones and it is independent of cyclic nucleotides. It can be classified as a serine kinase since the phosphorylation of the alpha subunit of eIF-2 is produced in serine residues. Under these conditions none of the kinase subunits are phosphorylated.     

Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1-IF2 interaction

Bacterial translation initiation factor IF1 is homologous to archaeal aIF1A and eukaryal eIF1A, which form a complex with their homologous IF2-like factors (aIF5B and eIF5B respectively) during initiation of protein synthesis. A similar IF1-IF2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30S-IF2 interaction by IF1. Here we compare Escherichia coli IF1 with thermophilic factors from Bacillus stearothermophilus and Thermus thermophilus. All three IF1s are structurally similar and functionally interchangeable in vivo and in vitro. However, the thermophilic factors do not stimulate ribosomal binding of IF2DeltaN, regardless of 30S subunits and IF2 origin. We conclude that an IF1-IF2 interaction is not universally conserved and is not essential for cell survival.     

Purification of membrane-bound ferredoxin: NADP+ oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids

A method is described for the isolation and purification of ferredoxin-NADP⁺ oxidoreductase (FNR, E.C. 1.18.1.2) and plastocyanin from spinach thylakoids. FNR is recovered from pools which are loosely and tightly bound to the membrane, with minimal disruption of pigment-protein complexes; yields can thus be higher than from procedures which extract only the loosely bound enzyme.Washed thylakoid membranes were incubated with the dipolar ionic detergent CHAPS (3-(3-cholamidopropyl-dimethylammonio)-1-propane-sulfonate). This provided an extract containing FNR and PC as its principal protein components, which could be rapidly separated from one another by chromatography on an anion-exchange column. FNR was purified to homogeneity (as judged from sodium dodecyl sulfate gel electrophoresis and the ratio between protein and flavin absorption maxima), using chromatography on phosphocellulose followed by batchwise adsorption to, and elution from hydroxylapatite. Plastocyanin was further purified on a Sephadex G-75 molecular sieve column.A typical yield, obtained in 3–4 days from 1 kg of deveined spinach leaves, was 7 mg of pure FNR (a single protein of M_r=37,000) and 3.5 mg of plastocyanin.Abbreviations CHAPS- 3-(3-cholamidopropyl-dimethylammonio)-1-propanesulfonate) - Chl- chlorophyll - FNR- ferredoxin-NADP⁺ oxidoreductase - Mops- 3-(N-morpholino) propanesulfonic acid - PC- plastocyanin - PMSF- phenylmethanesulfonylfluoride - SDS- sodium dodecyl sulfate - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tricine- N-tris (hydroxymethyl) methylglycine     

The dynamic cycle of bacterial translation initiation factor IF3

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.