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.

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.

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)

Purification of active Escherichia coli ribosome recycling factor (RRF) from an osmo-regulated expression system.

Ribosome release factor (RRF) from Escherichia coli was overproduced from an osmo-expression vector. More than 40% of cell protein was RRF after 6 h of induction. A purification scheme is described that produced 50 mg of RRF from an initial culture of 2 L. The recycling time for ribosomes synthesising the tripeptide fMet-Phe-Leu in vitro in the absence of RF3 was reduced from 40 to 15 s by the addition of purified 1.5 microM RRF.

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.

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.

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.

Changes in anterior chamber depth and angle-recession, late complications to ocular contusion.

Of 46 patients consecutively admitted to hospital for ocular contusion with hyphaema in 1964 and 1965, 30 patients were followed-up somewhat more than 10 years later in order to assess late complications. Angle-recession was observed in 17 (57%), while none had developed glaucoma. An average increase in depth of the anterior chamber of 0.06 mm in the affected eye was observed; this differences is highly significant with P less than 0.005. However, as these changes are small in individual patients, measurement of the increase in anterior chamber depth can only be a supplement to gonioscopy.

Traumatic hyphaema treated ambulatory and without antifibrinolytic drugs.

Forty-four patients referred consecutively over a period of one year for treatment of traumatic hyphaema have been treated ambulatory and with no drugs. Four patients developed secondary haemorrhage; three regained normal visual acuity. The frequency of secondary haemorrhage is not statistically different from that in two previously published patient groups treated with bed rest and with tranexamic acid, respectively.

Keratitis dendritica. An epidemiological investigation.

One hundred and seven outbreaks of dendritic keratitis have been registered by the ophthalmologists over a 2-year-period in a region with a population of approximately 446 000 persons. The incidence was found to be 12/100 000/year. The average age of our sample was 46.5 years with the same age distribution for males and females, and with a non-significant predominance of males. No seasonal variation of dendritic keratitis was observed. 50% of the patients had previously suffered from dendritic keratitis. In 25% of the patients the onset of the disease had been preceded by an infectious disease, while 6% were on local steroid therapy for non-dendritic eye diseases prior to the dendritic outbreak.

Secondary haemorrhage following traumatic hyphaema. A comparative study of conservative and tranexamic acid treatment.

Three out of 56 consecutively admitted patients with traumatic hyhaema, treated conservatively, developed secondary haemorrhage. Of the following 64 patients treated with tranexamic acid, none developed secondary haemorrhage. Statistical analysis, using chi2 test with Yate's correction showed no statistical difference with regard to the occurrence of secondary haemorrhage between the two groups.

Unilateral traumatic cataract in children.

A survey is presented of the course of unilateral traumatic cataract in 15 children of 8 years of age and below, admitted consecutively to the Ophthalmological Department of the Odense University Hospital over a period of 9 years. The follow-up examination revealed that four patients had visual acuity of more than 6/18, and of these two retained binocular single vision and used contact lenses. The size of the material does not permit any conclusive statements to be made. Thus it is impossible to select the patients who will have either a good or a poor final result from the treatment, with regard to vision and binocular single vision.

The effect of indomethacin 1% ophthalmic suspension on the pupil during extracapsular cataract surgery.

Fifty-six patients who underwent extracapsular cataract surgery were randomized in two groups with 28 patients in each group. Group 1 was treated with standard preoperative dilation regime and group 2 received in addition indomethacin 1% ophthalmic solution. Horizontal and vertical pupil diameter measurements were taken before capsulotomy, before exprimation of the lens nucleus and before lens implantation. The size of the pupil at the time of capsulotomy and at the time just before lens exprimation was greatest in the non-indomethacin treated group, but at the time of lens implantation the pupil of the indomethacin treated group was greatest. It is concluded that even though indomethacin seems to be able to inhibit surgically induced miosis, probably through its inhibitory effect on prostaglandin synthesis, the effect is only marginal and from a clinical point of view it cannot be recommended.

Pneumatic retinopexy. A long term follow-up study.

PURPOSE: We have analysed the results of 48 consecutive cases of rhegmatogenous retinal detachment treated with pneumatic retinopexy in a long term follow-up study. RESULTS: Thirty six of the original 48 patients were available to follow-up. We had an average follow-up period of 8.1 years and a 75% 7 year follow-up. Single operation success was achieved in 83% of patients. Redetachment occurred in 3 patients at 11, 30 and 33 months. Complications found included new breaks in 14%,, cystoid macular oedema in 8%,, and proliferative vitreoretinopathy in 3% of patients. No previously unreported complications were found in spite of the long follow-up time. Final anatomic success (after 1 or more operations) at 6 months post operatively was found in 97% of patients. At follow-up we found final anatomic success in all patients. CONCLUSION: The use of pneumatic retinopexy instead of scleral buckling is discussed. We recommend the consideration of pneumatic retinopexy in uncomplicated selected cases of rhegmatogenous retinal detachment.

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.

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain.

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.