The eubacterial protein synthesis inhibitor pulvomycin interacts with archaeal elongation factor 1α from Sulfolobus solfataricus

The effect of pulvomycin on the biochemical and fluorescence spectroscopic properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α), the functional analog of eubacterial EF-Tu, was investigated. The antibiotic was able to reduce in vitro the rate of protein synthesis however, the concentration of pulvomycin leading to 50% inhibition (173 μM) was two order of magnitude higher but one order lower than that required in eubacteria and eukarya, respectively. The effect of the antibiotic on the partial reactions catalysed by SsEF-1α indicated that pulvomycin was able to decrease the affinity of the elongation factor toward aa-tRNA only in the presence of GTP, to an extent similar to that measured in the presence of GDP. Moreover, the antibiotic produced an increase of the intrinsic GTPase catalysed by SsEF-1α, but not that of its engineered forms. Finally, pulvomycin induced a variation in fluorescence spectrum of the aromatic region of the elongation factor and its truncated forms. These spectroscopic results suggested that a conformational change of the elongation factor takes place upon interaction with the antibiotic. This finding was confirmed by the protection against chemical denaturation of SsEF-1α, observed in the presence of pulvomycin. However, a stabilising effect of the antibiotic directly on the protein in the complex could takes place.     

Exchange of free GTP with EF-1α·GDP complex promoted by a factor EF-1β from pig liver

Eukaryotic polypeptide chain elongation factor 1 (EF-1) has been resolved into two complementary factors, EF-1α and EF-1β, both of which were purified. Recently, we find that [³H] GDP bound to purified EF-1α is replaced by exogenous GTP rather slowly when the reaction is carried out at ionic strength optimal for polyphenylalanine synthesis. EF-1β stimulates the exchange of free GTP with EF-1α·GDP, indicating that the function of EF-1β is, at least in part, similar to that of bacterial EF-Ts.     

Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α

In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called ‘stalk’. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α.     

Translational arrest in hypoxic potato tubers is correlated with the aberrant association of elongation factor EF-1α with polysomes

Translation elongation factor EF-1 became stably associated with potato tuber polysomes at the onset of hypoxia, coincident with a sharp rise in lactate and decrease in tissue pH. This aberrant association of EF-1 with polysomes also occurred when aerobic tuber extracts were acidified in vitro. Upon resumption of protein synthesis, an increase in the steady-state levels of EF-1, and expression of an EF-1/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1 to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1.     

An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3?

An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-[35S]Met-tRNAfMet to ribosomes. In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-[35S]Met-tRNAfMET/poly(A,U,G)/ribosome complexes. Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet. Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3. The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor. Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits.     

In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-1α is associated with microtubules in carrot cells

Summary Evidence indicates that elongation factor-1 (EF-1), a ubiquitous and abundant protein factor involved in the first step of peptide elongation, is also associated with the cytoskeleton in a variety of organisms. Although the effects of these associations on EF-1's translational function have not been examined, the associations do appear to result in non-passive effects on the cytoskeleton. A carrot homolog of EF-1, pp 50, has been reported to interact with microtubules in vitro, inducing the formation of microtubule bundles that can be dissociated by Ca²⁺/calmodulin. The characterization of anti-pp 50 antibodies is reported here. Immunocytochemistry, using anti-pp 50 and anti-tubulin antibodies, was used to investigate the co-localization of pp 50 and microtubules in situ. In carrot protoplasts fixed after detergent lysis, at least a fraction of pp 50 appears to be associated with microtubules. Treatment of such protoplasts with amiprophos-methyl (APM) reduced both the presence of microtubules and the co-localizing pp 50-associated fluorescence. In taxol-treated protoplasts, increases in both microtubules and the colocalizing pp 50-associated fluorescence were observed. When carrot protoplasts were fixed prior to detergent extraction, confocal laser scanning microscopy likewise revealed co-localization. Furthermore, what is likely to be a fluorescence resonance energy transfer (FRET) between fluorochromes associated with anti-pp 50 and anti-tubulin reporters was observed, indicating that some pp 50 is intimately associated with microtubules. The in situ cytoarchitectural evidence is consistent with a function previously proposed for pp 50 based on in vitro experiments — that pp 50 is a plant microtubuleassociated protein (MAP) whose function can be modulated by a Ca²⁺/calmodulin signal transduction mechanism in plant cells.Abbreviations APM amiprophos-methyl - BSA bovine serum albumin - EF-1 elongation factor-1-alpha - FRET fluorescence resonance energy transfer - MAP microtubule-associated protein - PBS phosphate buffered saline - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Nucleotide sequence of a DNA region comprising the gene for elongation factor 1α (EF-1α) from the ultrathermophilic archaeotePyrococcus woesei: Phylogenetic implications

Summary The gene encoding elongation factor 1 (EF-1, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeotePyrococcus woesei was localized within aBglII fragment of chromosomal DNA. Sequence analysis showed that the EF-1 gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNA_ser (GGA). The three genes follow each other immediately in the order EF-1·S10·tRNA_ser after a putative promoter located 55 bp upstream of the EF-1 gene. Alignment of the derived EF-1 sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed thatPyrococcus EF-1 is highly homologous (89% identity) toThermococcus celer EF-1, both being strikingly more similar to eukaryotic EF-1 than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony-although not evolutionary parsimony-support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of thePyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation ofPyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

Quality control of Photosystem II under light stress – turnover of aggregates of the D1 protein in vivo

When photodamaged under excessive light, the D1 protein is digested and removed from Photosystem (PS) II to facilitate turnover of the protein. In vitro studies have shown that part of the photodamaged D1 protein forms aggregates with surrounding polypeptides before being digested by a protease(s) in the stroma [Yamamoto Y (2001) Plant Cell Physiol 42: 121–128]. The aim of this study was to examine whether light-induced aggregation of the D1 protein also occurs in vivo. The following results were obtained: (1) PS II activity in spinach leaves was significantly inhibited by weak illumination (light intensity, 20–100 μE m⁻² s⁻¹), as monitored by chlorophyll fluorescence Fv/Fm, when the leaves were kept at higher temperatures (35–40 °C); (2) aggregation of the D1 protein, as well as cleavage of the protein, was detected in thylakoids isolated from spinach leaves that had been subjected to heat/light stress; (3) aggregates of the D1 protein disappeared after incubation of the leaves at 25 °C in the dark or under illumination with weak light. Since it is dependent on the presence of oxygen, aggregation of the D1 protein is probably induced by reactive oxygen species produced in thylakoids upon illumination at elevated temperatures. Consistent with this notion, singlet oxygen production in thylakoid samples under illumination was shown to be stimulated significantly at higher temperatures.     

Rapid fold and structure determination of the archaeal translation elongation factor 1β from Methanobacterium thermoautotrophicum

The tertiary fold of the elongation factor, aEF-1, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1 was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1 structure revealed close similarity to its human analogue, eEF-1. In agreement with studies on EF-Ts and human EF-1, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1. aEF-1 was also found to bind calcium in the groove between helix 2 and strand 4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.     

Biochemical characterisation of the D60A mutant of the elongation factor 1α from the archaeon Sulfolobus solfataricus

The D60A mutant of the elongation factor (EF) 1α from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1α; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.     

Identification of an elongation factor 1Bγ protein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.     

Interactions of quinone with the iron-sulfur protein of the bc(1) complex: is the mechanism spring-loaded?

Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.