Protein Synthesis Elongation Factor Research Articles

Elongation factor-1 alpha mRNA is selectively translated following mitogenic stimulation.

Stimulation of quiescent Swiss 3T3 cells to proliferate leads to a selective 6-fold increase in the rate of translation of protein synthesis elongation factor eEF-1 alpha. Northern blot and solution hybridization protection studies show that levels of eEF-1 alpha mRNA remain constant following serum stimulation, demonstrating that eEF-1 alpha transcripts are not degraded following mitogenic stimulation and that the increase in eEF-1 alpha synthesis is accounted for by pre-existing mRNA. Localization of these transcripts in resting cells shows that they are largely distributed equally between stored mRNA protein particles and monosomes/disomes. Following serum addition, eEF-1 alpha transcripts present in mRNA protein particles redistribute to large polysomes rather than to monosomes and disomes as would be expected. The same is true for those transcripts present in monosomes and disomes. Salt-shift and translational runoff studies indicate that eEF-1 alpha transcripts sedimenting with monosomes and disomes in quiescent cells are associated with actively translating ribosomes. The results suggest that a specific transcript can move within polysome profile as a function of the affinity of translational apparatus for that transcript.

The use of protein synthesis elongation factor EF-Tu as internal calibration standard in two-dimensional electrophoretic studies of differentiation in Streptomyces.

Protein synthesis elongation factor EF-Tu is presented as an internal calibration standard for quantitative analysis of two-dimensional (2-D) protein electrophoresis gels. EF-Tu was selected on the basis of concentration measurements in cell-free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one-dimensional (1-D) and 2-D electrophoresis gels. The results demonstrated that the amount of EF-Tu synthesized in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF-Tu an ideal internal protein standard for quantitation of protein spots on 2-D electrophoresis gels. For such quantitative analysis we developed a computer-aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.

Purification and N-Terminal Sequence-Analysis of Pea Chloroplast Protein Synthesis Factor EF-G

Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea ( Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.

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

A cDNA clone (pBLT63) encoding a protein synthesis elongation factor 1 alpha (EF-1 alpha) 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 alpha plant genes from tomato and Arabidopsis. The barley genome contains an EF-1 alpha gene family situated on the short arm of chromosome 2 and the long arm of chromosome 5.

Altered expression and phosphorylation of amyloid precursor protein in heat shocked neuronal PC12 cells

The pathology of the Alzheimer's disease (AD) brain, including amyloid plaques, neurofibrillary tangles and neuronal degeneration, indicates that neurons affected by AD exist under conditions of stress. In fact, the brains of AD patients undergo many changes classically associated with the heat shock response, which is one form of a stress response. These changes include reduced protein synthesis, disrupted cytoskeleton, increased number of proteins associated with ubiquitin, and the induction of heat shock proteins. To investigate the response of neurons to stress, we examined neuronal PC12 cells incubated at either 37°C (control cells) or 45°C (heat-shocked cells). After a 30 min exposure at 45°C, the heat shocked cells exhibited several features characteristic of the classical heat shock response including a 45% reduction in total protein synthesis, the induction of heat shock protein 72, and an increased phosphorylation of the protein synthesis initiation factor eIF-2α. We used this cellular model system to study the neuronal response to stress specifically focusing on protein synthesis elongation factor 2 (EF-2) and the Alzheimer's amyloid precursor protein (APP), the precursor form of β-amyloid peptide. Hyperphosphorylation of EF-2 has been observed in the neocortex and hippocampus of AD brain 18. However, in our system, we find no hyperphosphorylation of EF-2 in response to heat shock. Heat-shocked neuronal PC12 cells exhibited two additional APP-like polypeptides not present in controls. We also found a significant decrease in the phosphorylation state of APP in response to heat shock. Protein kinase C phosphorylated APP in the neuronal PC12 cells, as evidenced by the response to PMA, a phorbol ester which stimulated the phosphorylation of APP and staurosporine which reduced its phosphorylation. Therefore, heat-shocked neuronal PC12 cells undergo several biochemical alterations which are also associated with the AD brain. We have used heat-shocked neuronal PC12 cells as a cell culture model system to investigate changes in several proteins (EF-2, APP, and tau) known to be altered in the AD brain.

Saccharomyces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin.

Protein synthesis elongation factor 2 (EF-2) is the target of the ADP-ribosylating activity of diphtheria toxin which is responsible for cell killing. Diphthamide, an unique post-translationally modified histidine residue, is both required for and the site of this ADP-ribosylation. Although present in the EF-2 of all eukaryotes and archaebacteria, the function of diphthamide is unknown. Here we describe the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 699, in yeast EF-2. Plasmid-borne EFT was randomly mutagenized at the histidine 699 codon, and the technique of plasmid shuffling was utilized to select strains that were maintained by the mutant EFT. These mutants were screened for diphtheria toxin resistance. Sequence analysis of the EFT in 49 toxin-resistant isolates showed that histidine 699 had been replaced by 1 of 4 amino acids: asparagine, glutamine, leucine, or methionine. All 11 of the possible codons corresponding to these 4 amino acids were found. The growth rates of cells sustained by the mutant forms of EF-2 were slightly slower than those of isogenic wild-type cells. We conclude that despite its strict conservation and universal post-translational modification, the histidine precursor of diphthamide is not essential to the function of yeast EF-2 in protein synthesis.

Sequence of a cDNA encoding the α-subunit of wheat translation elongation factor 1

A cDNA encoding the α-subunit of wheat protein synthesis elongation factor 1 (EF-1α) was isolated from a wheat cDNA expression library and sequenced. The deduced amino acid sequence is compared to EF-1α from other species and to elongation factor Tu (EF-Tu) from Escherichia coli. Putative GTP-binding sites are identified.

Inhibition of bacterial protein synthesis by elongation-factor-Tu-binding antibiotics MDL 62,879 and efrotomycin.

MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis. MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu). We prepared ribosomes and protein-synthesis elongation factors from three sources: E. coli, Bacillus subtilis, and a strain of B. subtilis selected for resistance to MDL 62,879 (strain G1674). Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B. subtilis and E. coli. We showed that in cell-free protein synthesis: (a) E. coli was sensitive to both MDL 62,879 and efrotomycin; (b) B. subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B. subtilis G1674 was resistant to both antibiotics. In the E. coli system and in the system from wild-type B. subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B. subtilis G1674. This demonstrates that the antibiotic acts by inhibition of EF-Tu. In contrast, extracts from B. subtilis failed to restore activity in an efrotomycin-inhibited E. coli system. Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action.

Membrane-associated GTPases in bacteria.

Members of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells. This review focuses on membrane-associated GTPases that have been described in prokaryotes. In bacteria, LepA and NodQ are very similar to protein synthesis elongation factors but apparently have membrane-related functions. The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion. Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability. In spite of similarities to well-studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated.

Synthesis of diphthamide: the target of diphtheria toxin catalyzed ADP-ribosylation in protein synthesis elongation factor 2

Synthesis of diphthamide: the target of diphtheria toxin catalyzed ADP-ribosylation in protein synthesis elongation factor 2

Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis

Mutations in the Bacillus subtilis major RNA polymerase sigma factor gene ( rpoD/ crsA47) and a sensory receiver gene ( spoOA/ rvtA11) are potent intergenic suppressors of several stage 0sporulation mutations ( spoOB, OE, OF & OK). We show here that these suppressors also rescue temperature-sensitive sporulation phenotypes (Spo ts) caused by mutations in RNA polymerase, ribosomal protein, and protein synthesis elongation factor EF-G genes. The effects of the crsA and rvtA suppressors on RNA polymerase and ribosomal protein spo ts mutations are similar to those previously described for mutations in another intergenic suppressor gene rev. We have examined the effects of rvtA and crsA mutations on the expression of sporulation-associated membrane proteins, including flagellin and penicillin binding protein 5 ∗ (PBP 5 ∗). Both suppressorsrestored sporulation and synthesis of PBP 5 ∗ in several spoO mutants. However, only rvtA restored flagelli synthesis in spoO suppressed backgrouds. The membrane protein phenotypes resulting from the presence of crsA or rvtA suppressors in spoO strainssuggests that these suppressors function via distinct molecular mechanism. The rvtA and crsA mutations are also able to block the ability of ethanol to induce spoO phenocopies at concentrations of ethanol which prevent sporulation in wild type cells. The effects of ethanol on sporulation-associated membrane protein synthesis in wild type and suppressor containing strains have been examined.

Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1 alpha genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1 alpha genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.

Altered sensitivity of eukaryotic elongation factor 2 for trypsin after phosphorylation and ribosomal binding

We have used variations in the trypsin sensitivity of eukaryotic protein synthesis elongation factor 2 (eEF-2) to probe for structural alterations induced by phosphorylation, ribosomal binding, or guanosine nucleotides. We could not detect any nucleotide-related effect on the tryptic cleavage rate of Arg66. However, eEF-2 was protected from trypsin after ribosomal binding. Also, phosphorylation of eEF-2 led to a protection of Arg66. This indicates that phosphorylation leads to a structural rearrangement that could explain the reduced affinity of the phosphorylated factor for ribosomes (Carlberg, U., Nilsson, A., and Nygård, O. (1990) Eur. J. Biochem. 191, 639-645). Cleavage of Arg66 led to a complete loss of the ability of the factor to be phosphorylated. Furthermore, ribosome-bound eEF-2 was found to be inaccessible for phosphorylation. Based on these findings and previously published data, we suggest that the region around the sites of phosphorylation and trypsin cleavage is vitally important for the factor function and ribosomal binding.

Transient foreign gene expression in polyethylene/glycol treated or electropulsated Eucalyptus gunnii protoplasts

Conditions for optimal transient gene expression of reporter genes (chloramphenicol acetyl transferase and β-glucuronidase) by polyethylene glycol or electrical treatment of Eucalyptus gunnii protoplasts derived from callus or cell suspension cultures were investigated. The effciency of electropermeabilisation depended on several factors including electrical parameters, pH and the source of protoplasts. Polyethylene glycol mediated DNA uptake was highly stimulated by heat shock pretreatment and was also more efficient than the electrical treatment. For both treatments the nature of the promoter associated with the reporter gene was very important. A promoter corresponding to a protein synthesis elongation factor gene was more effective than the classical promoter of the 35 S transcript from cauliflower mosaic virus.

Different fates of the chloroplast tufA gene following its transfer to the nucleus in green algae.

Previous work suggested that the tufA gene, encoding protein synthesis elongation factor Tu, was transferred from the chloroplast to the nucleus within the green algal lineage giving rise to land plants. In this report we investigate the timing and mode of transfer by examining chloroplast and nuclear DNA from the three major classes of green algae, with emphasis on the class Charophyceae, the proposed sister group to land plants. Filter hybridizations reveal a chloroplast tufA gene in all Ulvophyceae and Chlorophyceae and in some but not all Charophyceae. One charophycean alga, Coleochaete orbicularis, is shown to contain an intact but highly divergent chloroplast tufA gene, whose product is predicted to be non-functional in protein synthesis. We propose that a copy of the tufA gene was functionally transferred from the chloroplast to the nucleus early in the evolution of the Charophyceae, with chloroplast copies of varying function being retained in some but not all of the subsequently diverging lineages. This proposal is supported by the demonstration of multiple tufA-like sequences in Coleochaete nuclear DNA and in nuclear DNA from all other Charophyceae examined.

Role of lysine methylation in the activities of elongation factor 1α

Previous work in our laboratory has demonstrated that 19% of the lysine residues in the protein synthesis elongation factor (EF-1α) are methylated when the factor is purified from the mycelial form of the fungus Mucor racemosus. However, the same factor, when purified from spores of M. racemosus, is largely unmethylated. Despite its widespread occurrence in a great number of basic proteins, the functional significance of lysine N-methylation remains poorly understood. Spore and mycelial forms of EF-1α were therefore compared in a series of assays to determine their relative affinities for various substrates and cofactors known to interact with the factor during the elongation cycle. The results suggested that hypomethylated and fully methylated EF-1α had equal affinities for GTP, aminoacyl-tRNA, and ribosomes. Also, methylation did not appear to affect the accuracy of translation in an in vitro system. However, experiments did suggest that methylation may affect the ability of the factor to form complexes with other subunits (EF-1βγ) which are known to enhance the overall rate of protein synthesis.

Immunochemical Cross-Reactivities of Protein Synthesis Elongation Factors (EF-Tu and EF-1α Proteins) Support the Phylogenetic Coherence of Archaebacteria

Summary Elongation factor Tu (EF-Tu) proteins have been purified by affinity chromatography on GDP-Sepharose columns, from the eubacterium Thermotoga maritima and from archaebacteria (Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum) representative of all known divisions in the arachaebacterial tree except halophiles. Polyclonal antibodies raised against the purified Tu proteins were challenged with homologous and heterologous factors including eukaryotic EF-1α and cross-reactivities were quantified using 125 I labelled Protein A as the reporter molecule. The immunochemical relationships among factors both within and across kingdom boundaries demonstrated that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the analogous proteins of eubacteria and eukaryotes, (ii) within the archaebacteria the immunochemical cross-reactivities grossly correlate with the phylogenetic relatedness of the organisms inferred from similarities in rRNA sequences. The data support the notion that archaebacteria form a phylogenetically coherent taxon.

Bovine mitochondrial protein synthesis elongation factors: Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu. Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.

Content, distribution and stability of protein-synthesis elongation factor Tu in subcellular fractions of vegetative cells and spores ofStreptomyces aureofaciens

The protein synthesis elongation factor Tu (EF-Tu) was identified in dormant spores of Streptomyces aureofaciens and its content and distribution in vegetative cells and dormant spores were determined. Cell-free homogenates from spores were found to contain a EF-Tu cleaving membrane bound protease. The protease cleaved aggregated EF-Tu much less efficiently than non-aggregated factor in cell homogenates. The relative content of EF-Tu and ribosomes in dormant spores was very similar to that found in exponentially growing vegetative cells.

Content, distribution and stability of protein-synthesis elongation factor Tu in subcellular fractions of vegetative cells and spores of Streptomyces aureofaciens

The protein synthesis elongation factor Tu (EF-Tu) was identified in dormant spores of Streptomyces aureofaciens and its content and distribution in vegetative cells and dormant spores were determined. Cell-free homogenates from spores were found to contain a EF-Tu cleaving membrane bound protease. The protease cleaved aggregated EF-Tu much less efficiently than non-aggregated factor in cell homogenates. The relative content of EF-Tu and ribosomes in dormant spores was very similar to that found in exponentially growing vegetative cells.