Primary structure of elongation factor 1β from Artemia

cDNA complementary to mRNA coding for the elongation factor EF-1β has been cloned. A γgt 11 cDNA library has been screened with an antiserum against EF-1β which exchanges GDP bound to EF-1α with exogenous GTP during protein synthesis. The derived amino acid sequence corresponds to 208 amino acids including the N-terminal methionine which is absent in the mature protein. About sixty percent of the protein was sequenced by direct protein sequence analysis. Comparison of Artemis salina EF-1β with Escherichia coli EF-Ts shows no evident homology.     

Reductive activation of the methyl-tetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH

The enzymatic conversion of formaldehyde to CH₃S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH₃S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B_12a or B_12r to active B_12s.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - BES 2-bromoethanesulfonate - BCE boiled cell-free extract - DTT dithiothreitol - TCS 3,3,4,5-tetrachlorosalicylanilide - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - AMP-PNP 5-adenylyl imidophosphate     

Beyond translation: elongation factor-1α and the cytoskeleton

Summary While reported interactions of elongation factor-1 (EF-1) with various other molecules involved in protein biosynthesis are abundant, its interactions with major cytoskeletal proteins have not been as extensively examined. Major roles for EF-1 in cytoskeletal organization emerge from a review of such interactions within species as diverse as slime molds and mammals, sea urchins and higher plants. Based on these studies, the integration of EF-1's cytoskeletal roles with those of translation is considered, and prospective mechanisms for regulation of EF-1's cytoskeletal associations are discussed.Abbreviations EF elongation factor - RNP ribonucleoprotein particle - MT microtubule - MA mitotic apparatus - CaM calmodulin - MAP microtubule-associated protein     

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.     

A low-temperature-responsive translation elongation factor 1α from barley (Hordeum vulgare L.)

A cDNA clone (pBLT63) encoding a protein synthesis elongation factor 1 (EF-1) was isolated from a low-temperature winter barley shoot meristem library by differential screening. The nucleotide sequence of the coding region of the low-temperature-induced barley gene shows very high homology with two EF-1 plant genes from tomato and Arabidopsis. The barley genome contains an EF-1 gene family situated on the short arm of chromosome 2 and the long arm of chromosome 5. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Z23130.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Origin and early evolution of eukaryotes inferred from the amino acid sequences of translation elongation factors 1α/Tu and 2/G

Phylogenetic placements of archaebacteria and protozoa are important in understanding the origin and early evolution of eukaryotes. These problems have been analyzed mainly by comparisons of small subunit ribosomal RNA (SrRNA) sequences. However, the SrRNA phylogeny may sometimes be unreliable, especially when base compositions are biased among species. Because it is difficult to take full account of the bias in inferring the SrRNA tree, alternative examinations using protein sequence data have been very much desired. We analyzed the phylogenetic relationship among eukaryotes, archaebacteria, and eubacteria by the ML method of protein phylogeny using amino acid sequence data of EF-1α/Tu and 2/G. The unrooted tree analyses of both the EF-1α/Tu and 2/G consistently demonstrated that the ‘eocyte’ tree, in which archaebacteria are not monophyletic but eocytes are closer to eukaryotes than to other archaebacteria, is very likely. Further analysis using a composite tree of EF-1α/Tu and 2/G suggested that archaebacteria are closer to eukaryotes than to eubacteria but are not monophyletic. These results clearly support the hypothesis that eukaryotes have evolved from the eocyte-like organism. We also analyzed a protozoan phylogeny including mitochondrion-lacking species by the ML method using EF-1α and EF-2 data sets, and demonstrated (a) that two mitochondrion-lacking species, G. plecoglossi (Microsporidians) and G. lamblia (Diplomonads) probably represent the first and the second earliest offshoots of eukaryotes, respectively; (b) that Trypanosoma is not likely to have diverged next to Giardia as suggested by the SrRNA tree, but shows high affinity with higher eukaryotes; and (c) that protein phylogeny would give a robust estimation because amino acid compositions of conservative proteins do not differ significantly among species.     

Crystal structure of the read-through domain from bacteriophage Qβ A1 protein

Bacteriophage Qβ is a small RNA virus that infects Escherichia coli. The virus particle contains a few copies of the minor coat protein A1, a C‐terminally prolonged version of the coat protein, which is formed when ribosomes occasionally read‐through the leaky stop codon of the coat protein. The crystal structure of the read‐through domain from bacteriophage Qβ A1 protein was determined at a resolution of 1.8 Å. The domain consists of a heavily deformed five‐stranded β‐barrel on one side of the protein and a β‐hairpin and a three‐stranded β‐sheet on the other. Several short helices and well‐ordered loops are also present throughout the protein. The N‐terminal part of the read‐through domain contains a prominent polyproline type II helix. The overall fold of the domain is not similar to any published structure in the Protein Data Bank.     

The magic spot ppGpp influences in vitro the molecular and functional properties of the elongation factor 1α from the archaeon Sulfolobus solfataricus

Guanosine tetra-phosphate (ppGpp), also known as "magic spot I", is a key molecule in the stringent control of most eubacteria and some eukarya. Here, we show that ppGpp affects the functional and molecular properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α). Indeed, ppGpp inhibited archaeal protein synthesis in vitro, even though the concentration required to get inhibition was higher than that required for the eubacterial and eukaryal systems. Regarding the partial reactions catalysed by SsEF-1α the effect produced by ppGpp on the affinity for aa-tRNA was lower than that measured in the presence of GTP but higher than that for GDP. Magic spot I was also able to bind SsEF-1α with an intermediate affinity in comparison to that displayed by GDP and GTP. Furthermore, ppGpp inhibited the intrinsic GTPase of SsEF-1α with a competitive behaviour. Finally, the binding of ppGpp to SsEF-1α rendered the elongation factor more resistant to heat treatment and the analysis of the molecular model of the complex between SsEF-1α and ppGpp suggests that this stabilisation arises from the charge optimisation on the surface of the protein.     

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.     

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.     

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (K _i =150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme m, 2-(methylthio)ethanesulfonate - HS-HTP 7-mercaptoheptanoyl-l-threonine phosphate - CoM-SS-HTP the heterodisulfide of HS-CoM and HS-HTP - BCFE bolled cell-free extract - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - PEP phosphoenolpyruvate - 2,3-DPG 2,3-diphosphoglycerate - cDPG cyclic 2,3-diphosphoglycerate - 3-PG 3-phosphoglycerate - 2-PG 2-phosphoglycerate