Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli.

The C5aR antagonist (C5aRA)(1), which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli. To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37°C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496mg/L of NusA-fused C5aRA could be produced.

Posttranslational modification of Elongation Factor P from Staphylococcus aureus.

Antibiotic-resistant Staphylococcus aureus is becoming a major burden on health care systems in many countries, necessitating the identification of new targets for antibiotic development. Elongation Factor P (EF-P) is a highly conserved elongation protein factor that plays an important role in protein synthesis and bacteria virulence. EF-P undergoes unique posttranslational modifications in a stepwise manner to function correctly, but experimental information on EF-P posttranslational modifications is currently lacking for S. aureus. Here, we expressed EF-P in S. aureus to analyze its posttranslational modifications by mass spectrometry and report experimental proof of 5-aminopentanol modification of S. aureus EF-P.

Two forms of elongation factor 1 alpha (EF-1 alpha O and 42Sp50), present in oocytes, but absent in somatic cells of Xenopus laevis.

We have purified and partially sequenced the EF-1 alpha protein from Xenopus laevis oocytes (EF-1 alpha O). We show that the two cDNA clones isolated by Coppared et al. (Coppard, N. J., K. Poulsen, H. O. Madsen, J. Frydenberg, and B. F. C. Clark. 1991. J. Cell Biol. 112:237-243) do not encode 42Sp50, as claimed by these authors, but two very similar forms of EF-1 alpha O (EF-1 alpha O and EF-1 alpha O1). 42Sp50 is the major protein component of a 42S nucleoprotein particle that is very abundant in previtellogenic oocytes of X. laevis, 42Sp50 differs from EF-1 alpha O not only by its amino acid sequence, but also by several properties already reported. In particular, 42Sp50 has a low EF-1 alpha activity. It is distributed uniformly in the cytoplasm of previtellogenic oocytes, in contrast to EF-1 alpha O which is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body.

Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

Microtubule severing by elongation factor 1 alpha.

An activity that severs stable microtubules is thought to be involved in microtubule reorganization during the cell cycle. Here, a 48-kilodalton microtubule-severing protein was purified from Xenopus eggs and identified as translational elongation factor 1 alpha (EF-1 alpha). Bacterially expressed human EF-1 alpha also displayed microtubule-severing activity in vitro and, when microinjected into fibroblasts, induced rapid and transient fragmentation of cytoplasmic microtubule arrays. Thus, EF-1 alpha, an essential component of the eukaryotic translational apparatus, appears to have a second role as a regulator of cytoskeletal rearrangements.

Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome.

The 26S proteasome, composed of the 20S core and 19S regulatory complexes, is important for the turnover of polyubiquitinated proteins. Each subunit of the complex plays a special role in proteolytic function, including substrate recruitment, deubiquitination, and structural contribution. To assess the function of some non-essential subunits in the 26S proteasome, we isolated the 26S proteasome from deletion strains of RPN13 and RPN14 using TAP affinity purification. The stability of Gcn4p and the accumulation of ubiquitinated Gcn4p were significantly increased, but the affinity in the recognition of proteasome was decreased. In addition, the subcomplexes of the isolated 26S proteasomes from deletion mutants were less stable than that of the wild type. Taken together, our findings indicate that Rpn13p and Rpn14p are involved in the efficient recognition of 26S proteasome for the proteolysis of ubiquitinated Gcn4p.

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu.

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.

Resistance of archaebacterial aEF-1 alpha.GDP against denaturation by heat and urea.

The elongation factor aEF-1 alpha, isolated as aEF-1 alpha.GDP from the thermoacidophilic archaebacterium Sulfolobus solfataricus, exchanges GDP for [3H]GDP at a rate which reaches a maximum at 95 degrees C. The rate constants at different temperatures of the heat inactivation of aEF-1 alpha.GDP are considerably lower compared to those referred to Escherichia coli EF-Tu.GDP. The Tm values determined for both aEF-1 alpha.GDP and EF-Tu.GDP are 97 and 53 degrees C, respectively. The addition of GDP during the heat treatment protects significantly EF-Tu.GDP but only slightly aEF-1 alpha.GDP. The ability of aEF-1 alpha.GDP to exchange GDP for [3H]GDP is impaired at 70 degrees C by urea at concentrations which are greater compared to those required to inactivate E. coli EF-Tu.GDP at 45 degrees C; apparently both factors are not protected by GDP against inactivation by urea.

Isolation, crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes.

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.

Archaeal elongation factor 1 beta is a dimer. Primary structure, molecular and biochemical properties.

The elongation factor 1 beta (EF-1 beta), that in eukarya and archaea promotes the replacement of GDP by GTP on the elongation factor 1 alpha x GDP complex, was purified to homogeneity from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1 beta). Its primary structure was established by sequenced Edman degradation of the entire protein or its proteolytic peptides. The molecular weight of SsEF-1 beta was estimated as about 10000 or 20000 under denaturing or native conditions respectively; this finding suggests that the native protein exists as a dimer. The peptide chain of SsEF-1 beta is much shorter than that of its eukaryotic analogues and homology is found only at their C-terminal region; no homology exists between SsEF-1 beta and eubacterial EF-Ts. At 50 degrees C, at a concentration of SsEF-1 beta 5-fold higher than that of SsEF-1 alpha x [3H]GDP the rate of the exchange of [3H]GDP for GTP becomes about 160-fold faster. An analysis of the values of the energetic parameters indicates that in the presence of SsEF-1 beta the GDP/GTP exchange is entropically favoured. At 100 degrees C the half-life of SsEF-1 beta is about 4 h.

Modification of the reactivity of three amino-acid residues in elongation factor 2 during its binding to ribosomes and translocation.

The accessibility of three amino acids of EF-2, located within highly conserved regions near the N- and C-terminal extremities of the molecule (the E region and the ADPR region, respectively) to modifying enzymes has been compared within nucleotide-complexed EF-2 and ribosomal complexes that mimic the pre- and posttranslocational ones: the high-affinity complex (EF-2)-nonhydrolysable GTP analog GuoPP[CH2]P ribosome and the low-affinity (EF-2)-GDP-ribosome complex, EF-2 and ribosomes being from rat liver. We studied the reactivity of two highly conserved residues diphthamide-715 and Arg-66, to diphtheria-toxin-dependent ADP-ribosylation and trypsin attack, and of a threonine that probably lies between residues 51 and 60, to phosphorylation by a Ca2+/calmodulin-dependent protein kinase. Diphthamide 715 and this threonine residue were unreactive within the high-affinity complex but seemed fully reactive in the low-affinity complex. Arg-66 was resistant to trypsin in both complexes. The possible involvement of the E and ADPR regions of EF-2 in the interaction with ribosome in the two complexes is discussed.

Site-directed mutants of post-translationally modified sites of yeast eEF1A using a shuttle vector containing a chromogenic switch.

Eukaryotic elongation factor 1A (eEF1A, formerly eEF-1 alpha) carries aminoacyl-tRNAs into the A-site of the ribosome in a GTP-dependent manner. In order to probe the structure/function relationships of eEF1A, we have generated site-directed mutants using a modification of a highly versatile yeast shuttle vector, which consists of the insertion of a 66 base long synthetic DNA fragment in the vector's polylinker. Via oligonucleotide-directed mutagenesis, the modification permits the identification of mutant clones based on a chromogenic screen of beta-galactosidase activity. Mutagenesis reactions are performed with two or more oligonucleotides, one introducing the chromogenic shift, and the other(s) introducing the mutation(s) of interest in eEF1A. Several rounds of chromogenic shifts and additional mutations can be performed in succession on the same vector. To address the possible function of the methylated lysines in yeast eEF1A, we have changed the post-translationally modified lysines (residue 30, 79, 316 and 390) to arginines using the above methodology. Yeast with eEF1A mutants that substitute arginine in all four sites do not show any phenotypic change. There is also an apparent equivalency of wild-type and mutant yeast eEF1A in in vitro assays. It is concluded that the post-translational modifications of eEF1A are not of major importance for eEF1A's role in translation.

Distinct functions of N and C-terminal domains of GreA, an Escherichia coli transcript cleavage factor.

The prokaryotic transcription factors GreA and GreB are involved in the regulation of transcript elongation by RNA polymerase (RNAP). Their known activities include suppression of transcription arrest, enhancement of transcription fidelity, and facilitation of the transition from abortive initiation to productive elongation. Presumably, Gre proteins exert their functions by altering the conformation of the enzyme in ternary elongation complexes (TEC) and inducing the cleavage of nascent RNA. GreA and GreB have a similar structural organization and consist of two domains: a C-terminal globular and an extended N-terminal coiled-coil domain. To investigate the functional roles of Gre domains, we expressed separately the N and C-terminal domains of GreA (NTD and CTD, respectively) and characterized their activities with in vitro assays. We demonstrate that the NTD possesses the residual transcript cleavage activity of the wild-type GreA. The CTD does not display any nucleolytic activity; however, it substantially increases the cleavage activity of the NTD. In contrast to NTD, the CTD competes with GreA and GreB for binding to RNAP and inhibits their transcript cleavage and antiarrest activities. Both domains individually and together inhibit transcription elongation. From these results we conclude that the NTD is responsible for the GreA induction of nucleolytic activity while the CTD determines the binding of GreA to RNAP. Both domains are required for full functional activity of GreA.

Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA.

In bacteria, the selenocysteine-specific elongation factor SelB is necessary for incorporation of selenocysteine, the 21st amino acid, into proteins by the ribosome. SelB binds to an mRNA hairpin formed by the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA (Sec-tRNASec) at the ribosomal A site. The minimum fragment (residues 512-634) of Moorella thermoacetica SelB (SelB-M) required for mRNA binding has been overexpressed and purified. The complex of SelB-M with 23 nucleotides of the SECIS mRNA hairpin was crystallized at 293 K using the hanging-drop vapour-diffusion or oil-batch methods. The crystals diffract to 2.3 A resolution using SPring-8 BL41XU and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 81.69, b = 169.58, c = 71.69 A.

Heterogeneity of native rat liver elongation factor 2.

The high heterogeneity of native rat liver EF-2 prepared from either 105000 x g supernatant or microsome high-salt extract was detected by two-dimensional equilibrium isoelectric focusing-SDS-polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of Mr 95,000, which were not artefactual for their amount varied when EF-2 was specifically ADP-ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin-dependent protein kinase (most likely Ca2+/calmodulin-dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299-17303; (1988) Nature 334, 170-173]). Results of ADP-ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP-ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF-2. Phosphorylated and ADP-ribosylated forms of EF-2 could be ADP-ribosylated and phosphorylated, respectively, but a native form both ADP-ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF-2 and of its phosphorylated and ADP-ribosylated derivatives.

[Cellular distribution of elongation factor EF1 in wheat germ]. / Distribution cellulaire des facteurs d'élongation EF1 du germe de blé

Cellular distribution of elongation factors (EF1) from imbibed then redessicated wheat embryos is determined after purification and analytical gel electrophoresis of soluble and ribosome-bound factors. Two heavy forms (EF1 H, mol. wt, 250 000) are found in cytosol while ribosome-bound factors contain a light form (EF1L, mol. wt, 45 000) with the greatest activity and a heavy form (mol. wt, 160 000) which might well be an intermediary in the recycling of ribosomal factor EF1L to soluble factor EF1H.

Isolation and characterization of a rice cDNA encoding the gamma-subunit of translation elongation factor 1B (eEF1Bgamma).

We isolated a rice cDNA clone (refg) encoding the gamma-subunit of translation elongation factor 1B (eEF-1B gamma; the old designation was EF-1 gamma). The refg encodes an open reading frame of 419 amino acids which shows a similarity to the equivalent sequences from animals and yeast. Complex formation analysis, which showed the recombinant protein of refg (His-eEF1B gamma) and formed a complex with GST-eEF-1Bbeta, indicated that the refg encodes rice eEF1B gamma of the eEF1B alphabeta gamma complex. Expression analysis showed that refg mRNA is very abundant in suspension-cultured cells during the exponential phase of growth. A DNA blot analysis indicated that refg is located at a single locus in the rice genome.

A new translational elongation factor for selenocysteyl-tRNA in eucaryotes.

In eucaryotes, selenocysteine (SeCys) was found in some selenoproteins, but SeCys-tRNA was not recognized by EF-1 alpha. A different translational elongation factor for SeCys-tRNA must therefore supply SeCys-tRNA to the machinery of selenoprotein translation. I found this factor in bovine liver extracts with a UGA-programmed ribosome binding assay. The activity of binding of [75Se]SeCys-tRNA to the UGA-programmed ribosomes was eluted in fractions 57-65 using a CM-Sephadex C-25 column, and separated from EF-1 alpha (the activity of binding of [14C]Phe-tRNA to the UUU-programmed ribosomes) in fractions 25-37. EF-1 alpha in fraction 25 could discriminate (UUU)10 for [14C]Phe-tRNA. A factor in fraction 57 could discriminate (UGA)10 for [75Se]SeCys-tRNA. The elution pattern of activity of binding of [75Se]SeCys-tRNA to the UGA-programmed ribosomes was almost identical to that of activity of protecting [75Se]SeCys-tRNA against alkaline hydrolysis (SePF activity) [FEBS Lett. 347 (1994) 137-142]. These two activities might depend on the same factor. The activity of binding of [75Se]SeCys-tRNA to the UGA-programmed ribosomes directly indicates that a factor in fraction 57 is a new translational elongation factor for SeCys-tRNA in eucaryotes.

A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-1 gamma and EF-1 beta.

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF-1 beta and EF-1 gamma. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.