Remarkable conservation of translation initiation factors: IF1/eIF1A and IF2/eIF5B are universally distributed phylogenetic markers.

Initiation of protein biosynthesis is an essential process occurring in cells throughout the three phylogenetic domains, Bacteria, Archaea, and Eucarya. IF1/eIF1A and IF2/eIF5B, two conserved translation initiation factors are involved in this important step of protein biosynthesis. The essentiality, universal distribution, conservation, and interspecies functional homology of both factors are a unique combination of properties ideal for molecular phylogenetic studies as demonstrated by the extensively compared SSU rRNAs. Here, we assess the use of IF1/eIF1A and IF2/eIF5B in universal and partial phylogenetic studies by comparison of sequence information from species within all three phylogenetic domains and among closely related strains of Haemophilus parainfluenzae. We conclude that the amino acid sequence of IF1/eIF1A-IF2/eIF5B is a universal phylogenetic marker and that the nucleotide sequence of the IF2/eIF5B G-domain is more credible than SSU rRNA for the construction of partial phylogenies among closely related species and strains. Because of these two application levels, IF1/eIF1A-IF2/eIF5B is a phylogenetic "dual level" marker.

Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county.

During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacterfreundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial sequencing of the gene encoding translation initiation factor 2. Fourteen of the 15 isolates were identical, as evaluated by their antibiograms and by all these typing methods. This epidemic strain harboured the aminoglycoside resistance genes aac(3)-II and ant(3")-I, with the latter located in tandem with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission.

Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature.

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.

Macromolecular mimicry in translation initiation: a model for the initiation factor IF2 on the ribosome.

Protein biosynthesis in bacteria is controlled by a number of translation factors. Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A-site-bound tRNA. This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2. Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex.

Identification of Enterobacteriaceae by partial sequencing of the gene encoding translation initiation factor 2.

Nucleotide sequence analysis is increasingly being used to identify bacteria. In this work, a PCR assay based on degenerate primers was used to obtain the partial sequence of infB, the gene encoding translation initiation factor 2 (IF2), in 39 clinical isolates of different Enterobacteriaceae. The partial sequence encodes the GTP-binding domain of IF2. Together with sequences from the literature, a total of 15 species, each represented by one to seven strains, was investigated. Phylogenetic analysis yielded an evolutionary tree which had a topology similar to a tree constructed using available 16S rRNA sequences. It is concluded that the inter-species variation of the infB gene fragment is sufficient for its use in the characterization of strains that have aberrant phenotypic reactions.

Characterization of the domains of E. coli initiation factor IF2 responsible for recognition of the ribosome.

We have studied the interactions between the ribosome and the domains of Escherichia coli translation initiation factor 2, using an in vitro ribosomal binding assay with wild-type forms, N- and C-terminal truncated forms of IF2 as well as isolated structural domains. A deletion mutant of the factor consisting of the two N-terminal domains of IF2, binds to both 30S and 50S ribosomal subunits as well as to 70S ribosomes. Furthermore, a truncated form of IF2, lacking the two N-terminal domains, binds to 30S ribosomal subunits in the presence of IF1. In addition, this N-terminal deletion mutant IF2 possess a low but significant affinity for the 70S ribosome which is increased by addition of IF1. The isolated C-terminal domain of IF2 has no intrinsic affinity for the ribosome nor does the deletion of this domain from IF2 affect the ribosomal binding capability of IF2. We conclude that the N-terminus of IF2 is required for optimal interaction of the factor with both 30S and 50S ribosomal subunits. A structural model for the interaction of IF2 with the ribosome is presented.

Immunochemical determination of cellular content of translation release factor RF4 in Escherichia coli.

The biosynthesis of proteins in prokaryotes is terminated when a stop codon is present in the A-site of the 70S ribosomal complex. Four different translation termination factors are known to participate in the termination process. Release factor RF1 and RF2 are responsible for the recognition of the stop codons, and RF3 is known to accelerate the overall termination process. Release factor RF4 is a protein involved in the release of the mRNA and tRNA from the ribosomal complex. Furthermore, RF4 is involved in the proofreading in the elongation step of protein biosynthesis. The cellular contents of RF1, RF2, and RF3 were determined earlier. Here we report the cellular content of RF4 in Escherichia coli to be approximately 16,500 molecules per cell. The cells were grown in a rich medium and harvested in the beginning of the exponential growth phase. The quantifications were performed by using Western immunoblotting with radioactive iodinated streptavidin and biotinylated rabbit anti-mouse immunoglobulins plus a highly specific monoclonal antibody against RF4 as first antibody.

A six-domain structural model for Escherichia coli translation initiation factor IF2. Characterisation of twelve surface epitopes.

The Escherichia coli translation initiation factor IF2 is a 97 kDa protein which interacts with the initiator fMet-tRNAfMet, GTP and the ribosomal subunits during initiation of protein biosynthesis. For structural and functional investigations of the factor, we have raised and characterised monoclonal antibodies against E. coli IF2. Twelve epitopes have been localised at the surface of the protein molecule by three different methods: Interactions of the monoclonal antibodies with nested deletion mutants of IF2, comparison of the relative location of the epitopes in a competition immunoassay and cross-reactivity analyses of the monoclonal antibodies towards IF2 from Salmonella typhimurium, Klebsiella oxytoca, Enterobacter cloacae, Proteus vulgaris, and Bacillus stearothermophilus. These data are combined with predicted secondary structure and discussed in relation to a six-domain structural model for IF2. The model describes IF2 as a slightly elongated molecule with a structurally compact C-terminal domain, a well-conserved central GTP-binding domain, and a highly charged, solvent exposed N-terminal with protruding alpha-helical structures.

Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits: a functional role for the N-terminus of the factor.

In the initiation step of bacterial protein synthesis initiation factor IF2 has to join the 30S ribosomal subunit in order to promote the binding of the fMet-tRNAMetf. In order to identify regions within IF2 which may be involved in the primary ribosome-factor interaction, we have constructed several C-terminal and N-terminal truncated forms of the factor as well as isolated structural domains, and tested them in a 30S ribosomal binding assay in vitro. Monoclonal antibodies with epitopes located within the two N-terminal domains of IF2 were used in these experiments. Hitherto, no function has been allocated to the N-terminal region of IF2. Here we show that a mutant consisting of the two N-terminal domains has intrinsic affinity to the ribosomal subunit. Furthermore, a deletion mutant of IF2 which is lacking the two N-terminal domains shows negligible affinity. Moreover mAb with epitopes located within domain II strongly inhibits the binding capacity of IF2 to the 30S ribosomal subunit, whereas mAb with epitopes mapped within domain I do not affect the binding of the factor. The C-terminal domain of IF2 shows no affinity for the small ribosomal subunit. In addition, mutants with C-terminal deletions are not significantly affected in this interaction. Therefore, we conclude that the N-terminus of IF2 has affinity per se to bind the ribosomal subunit, with domain II being directly involved in the interaction.

Immunochemical determination of the cellular content of polypeptide chain release factor RF3 in Escherichia coli.

Polypeptide chain termination in Escherichia coli is known to require two codon specific release factors, RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract; a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.

E. coli translation initiation factor IF2--an extremely conserved protein. Comparative sequence analysis of the infB gene in clinical isolates of E. coli.

The functionally uncharacterised N-terminal of translation initiation factor IF2 has been found to be extremely variable when comparing different bacterial species. In order to study the intraspecies variability of IF2 the 2670 basepairs nucleotide sequence of the infB gene (encoding IF2) was determined in 10 clinical isolates of E. coli. The N-terminal domains (I, II and III) were completely conserved indicating a specific function of this region of IF2. Only one polymorphic position was found in the deduced 890 amino acid sequence. This Gln/Gly490 is located within the central GTP/GDP-binding domain IV of IF2. The results are further evidence that IF2 from E. coli has reached a highly defined level of structural and functional development.

Osmo-expression and fast two-step purification of Escherichia coli translation termination factor RF-3.

The gene for the translation termination factor RF-3 in Escherichia coli has recently been cloned and sequenced. Only small amounts of the protein have been purified until now, not sufficient for detailed investigation of the structure and function of this factor. For such studies, we have developed an overexpression system and a purification procedure suitable for large quantities of RF-3. The gene prfC was cloned into the osmo-inducible plasmid pOSEX3 and subsequently transformed into the E. coli strain MKH13. The expression of prfC in this plasmid, which is under the control of the osmotic pressure in the growth medium, leads to a level of RF-3 more than 100-times higher than that in wild-type cells. Using a new two-step FPLC protein purification procedure consisting of ion-exchange chromatography on Q-Sepharose FF and S-Sepharose HP, we obtain 220 mg pure RF-3 from 10 g overproducing cells, corresponding to 55 mg RF-3/l medium. The identity of the purified protein was confirmed by matrix-assisted laser desorption/ionisation mass spectrometry of tryptolytic fragments and by N-terminal amino acid sequencing. The activity of the purified factor was tested in vitro by measuring the stimulation of RF-2 dependent formylmethionine release from a ribosomal termination complex and the binding capacity of GTP and GDP. All assays showed that the purified RF-3 was highly active with a specific activity of approximately 2000 units/mg.

Improved recombinant tandem expression of translation initiation factor IF2 in RNASE E deficient E. coli cells.

The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2 alpha, IF2 beta and IF2 gamma differing only in the N-terminal: IF2 beta and IF2 gamma lack 158 and 165 amino acid residues, respectively, as compared to IF2 alpha. We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2 alpha, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2 beta and IF2 gamma is dependent on or related to a posttranscriptional processing of the polycistronic nusA operon, containing infB, the gene for IF2. Here we have used S1 mapping to study the existence of such mRNA processing in the region between the initiation sites for IF2 alpha and IF2 beta/IF2 gamma. The results show a Ribonuclease E cleavage site at position +200 in the infB mRNA between the translation initiation sites. However, studies of the overexpression of the different forms of IF2 show that the relative expression of IF2 alpha and IF2 beta/IF2 gamma is independent of RNase E activity. Thus E. coli exhibits a true tandem translation of intact infB mRNA with multiple in-frame translation initiation sites resulting in gene products of different sizes. An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E activity. We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated, resulting in an elevated amount of recombinant IF2. This observation may have a more general application within the field of recombinant protein production and expression efficiency.

Protease activity of outer membrane protein OmpT in clinical E.coli isolates--studies using translation initiation factor IF2 as target protein.

During purification of the translation initiation factor IF2 from ompT+ strains of Escherichia coli the IF2 is partially degraded in the presence of membrane debris during the first steps of purification. This is a result of proteolytic cleavage by outer membrane protease OmpT [1]. Here we have investigated the activity of OmpT in 51 clinical E. coli isolates of human origin, by a time dependent OmpT activity assay using IF2 as target protein. The activity of OmpT in the outer cell membrane is highly variable among wild type E.coli strains, ranging from no detectable activity in 65% of the strains to a very high activity in 5 strains (10%). The OmpT activity is closely related to the assay temperature and to the growth temperature of the cells, and can be reduced or even eliminated by lowering the temperature of growth. The results open the possibility of using non-denaturing gel electrophoresis of crude cell lysates as a screening method in population genetic studies of initiation factor IF2 and other cytoplasmic proteins which are cleaved by OmpT.

The reference range for complexed alpha 2-macroglobulin human plasma: development of a new enzyme linked in immunosorbent assay (ELISA) for quantitation of complexed alpha 2-macroglobulin.

Purified alpha 2-macroglobulin was complexed by reaction with methylamine and used to raise monoclonal murine antibodies. A four-step enzyme linked immunosorbent assay (ELISA) was developed to determine the antibody-specificity of the produced monoclonal murine antibodies towards human native and complexed alpha 2-macroglobulin. Two monoclonal antibodies were selected, H11A11 (specific towards complexed alpha 2-macroglobulin) and 1CG4 (recognizes both forms of the molecule), and purified by affinity chromatography on protein G. The purified antibodies were used to develop a fast three-step ELISA for exact quantitation of complexed and total alpha 2-macroglobulin in human plasma. The intra-assay coefficient of variation (CV) for measurement of complexed alpha 2-macroglobulin is 2.2-9.9%, whereas the inter-assay CV was determined to be 3.7-10.5% and the recovery of the assay is 93-108%. The assay for total alpha 2-macroglobulin has an intra-assay CV of 3.0-15.5%, an interassay CV of 5.1-21.2% and a recovery of 91-116%. Citrated plasma samples from 139 healthy blood donors were examined, resulting in a reference range for complexed alpha 2-macroglobulin of 13.5-31.1 mg 1(-1) with a median value of 21.7 mg 1(-1). The concentration of total alpha 2-macroglobulin was measured by the same assay using the monoclonal antibodies 1CG4. For total alpha 2-macroglobulin we determined the reference range to be 1.12-3.54 g 1(-1) with a median value of 2.14 g 1(-1). Based on these results the reference range for complexed alpha 2-macroglobulin as a percentage of total alpha 2-macroglobulin was calculated to be 0.8-1.9% with a median value of 1.0%.

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.

OmpT proteolysis of E. coli initiation factor IF2. Elimination of a cleavage site by site-directed mutagenesis.

A serious problem during purification of the E. coli initiation factor IF2 is a significant loss of native IF2 due to partial degradation. We have previously shown that the major fragment, IF2 gamma (65 kDa), is the result of cleavage of IF2 alpha at the peptide bond between lysine 289 and arginine 290. In this paper we demonstrate that the cleavage is a result of proteolysis by outer membrane protease OmpT occurring immediately after cell lysis and in the S30 supernatant. By protein engineering we have constructed an IF2 mutant Lys289-greater than Met and shown that the IF2 gamma cleavage site in this mutant protein is insensitive to cleavage by OmpT. However the mutant protein is cleaved by OmpT between arginine 279 and alanine 280, which is a novel sequence specificity for this protease.

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.

Structural and functional domains of E coli initiation factor IF2.

Initiation of translation in prokaryotes requires the participation of at least three soluble proteins: the initiation factors IF1, IF2 and IF3. Initiation factor 2, which is one of the largest proteins involved in translation (97.3 kDa) has been shown to stimulate in vitro the binding of fMet-tRNA(fMet) to the 30S ribosomal subunit. After formation of 70S translation initiation complex, IF2 is believed to participate in GTP hydrolysis, thereby promoting its own release. Here we review evidence which indicates the functional importance of the different structural domains of IF2, emphasizing new information obtained by in vivo experiments.

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)