Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1 [alpha]. Search for a function

The 3'-terminal tRNA-like structure in turnip yellow mosaic virus (TYMV) RNA can be adenylated by tRNA nucleotidyltransferase and subsequently aminoacylated by valyl-tRNA synthetase.Here we present evidence that TYMV Val-RNA can form a stable complex with eukaryotic wheat germ elongation factor EF-1alpha and GTP: the Val-RNA is protected by EF-1alpha.. GTP against digestion by RNase A. By affinity chromatography of TYMV Val-RNA fragments on immobilized EF-1alpha . GTP, it has been established that the valylated aminoacyl RNA domain, which in TYMV RNA is formed by the 3' half of the tRNA-like region, is sufficient for complex formation with EF-1alpha . GTP. The aminoacyl RNA domain is equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop. In line with these results, the aminoacyl RNA domain in TYMV Val-RNA complexed to EF-1 alpha . GTP is resistant to digestion by RNase A. It is also shown that the TYMV RNA replicase (RNA-dependent RNA polymerase) isolated from TYMV-infected Chinese cabbage leaves does not contain tRNA nucleotidyltransferase, valyl-tRNA synthetase or EF-1alpha. This suggests that interaction of TYMV RNA with EF-1alpha is not mandatory for replicase activity.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.     

Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated nuclear localization

Eukaryotic polypeptide elongation factor EF-1 is not only a major translational factor, but also one of the most important multifunctional (moonlighting) proteins. EF-1 consists of four different subunits collectively termed EF-1alphabeta beta'gamma and EF-1alphabeta gammadelta in plants and animals, respectively. EF-1alpha x GTP catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome. EF-1beta beta'gamma (EF-1beta and EF-1beta'), catalyzes GDP/GTP exchange on EF-1alpha x GDP to regenerate EF-1alpha x GTP. EF-1gamma has recently been shown to have glutathione S-transferase activity. EF-2 catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site on the ribosome. Recently, molecular mimicry among tRNA, elongation factors, releasing factor (RF), and ribosome recycling factor (RRF) has been demonstrated and greatly improved our understanding of the mechanism of translation. Moreover, eukaryotic elongation factors have been shown to be concerned or likely to be concerned in various important cellular processes or serious diseases, including translational control, signal transduction, cytoskeletal organization, apoptosis, adult atopic dermatitis, oncogenic transformation, nutrition, and nuclear processes such as RNA synthesis and mitosis. This article aims to overview the recent advances in protein biosynthesis, concentrating on the moonlighting functions of EF-1.     

A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation factor-1 alpha.

Higher plant proteins immunologically related to the animal substrate adhesion molecule vitronectin have recently been observed and implicated in a variety of biological processes, such as plasma membrane-cell wall adhesion, pollen tube extension, and bacterium-plant interaction. We provide evidence that, similar to vitronectin, one of these proteins, PVN1 (plant vitronectin-like 1), isolated from 428 mM NaCl-adapted tobacco cells binds to glass surfaces an heparin. PVN1 was isolated by glass bead affinity chromatography. Isolated PVN1 has adhesive activity based on results from a baby hamster kidney cell-spreading assay. This plant adhesion protein was detected in all tissues examined but was most abundant in roots and salt-adapted cultured cells. Immunogold labeling indicated that PVN1 is localized in the cell wall of cortical and transmitting tissue cells of pollinated mature styles. A partial amino acid sequence of PVN1 revealed no similarity with vitronectin but, instead, was nearly identical to the translational elongation factor-1 alpha (EF-1 alpha). A clone isolated by screening a tobacco cDNA expression library with anti-PVN1 encoded a protein with greater than 93% identity to sequences of EF-1 alpha from plants of numerous species. Immunological cross-reactivity between tobacco PVN1 and EF-1 alpha as well as the reaction between the EF-1 alpha antibody and the 65- and 75-kD vitronectin-like proteins of a fucoidal alga supported the conclusion that the plant extracellular adhesion protein PVN1 is related to EF-1 alpha.     

In Vitro Replication of Cowpea Mosaic Virus RNA III. Template Recognition by Cowpea Mosaic Virus RNA Replicase

Cowpea mosaic virus (CPMV) RNA replicase has been purified about 200-fold from CPMV-infected Vigna unguiculata leaves. Optimal reaction conditions for replicase activity have been established that allow RNA synthesis to proceed for at least 15 h. Using a polymerase assay under conditions optimal for CPMV RNA-directed RNA synthesis, all natural RNA species tested appeared to be able to direct the incorporation of labeled ribonucleotides, whereas synthetic homoribopolymers were either inactive or only slightly active. Using a nitrocellulose membrane filter assay to measure complex formation between the replicase preparation and various RNA species, all natural RNA species tested, except that of the comovirus radish mosaic virus, appeared to be unable to compete with ³²P-labeled CPMV RNA in binding to replicase. We propose that CPMV replicase is actually template specific but does not display this property in a polymerase assay, since labile complexes between heterologous templates and replicase become stabilized by the formation of phosphodiester bonds. From homoribopolymer competition binding experiments we conclude that the polyadenylic acid on the CPMV genome might be a part of the replicase binding site.     

Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (M_r > 500,000) containing four distinct polypeptides (α, β, γ, δ). The other form, EF-1α, consists of a single polypeptide which is the same as the α subunit of EF-1H. Both EF-1α and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1α is enhanced in polyphenylalanine synthesis by a complementary component, EF-1βδ. It is also shown that EF-1βδ can facilitate an exchange of EF-1α-bound GDP for GTP. The EF-1α dissociation constants for GDP and GTP were 0.47 and 0.55 μm respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 μm, respectively. Thus, while EF-1α and EF-1H had approximately the same affinities for GDP and GTP, the EF-1α dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1α or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.     

A poly(U)-binding factor stimulating EF-1 activity in the wheat-germ soluble fraction

A poly(U)-binding activity is present in the high-speed supernatant fraction of embryo homogenates from wheat seeds. The factor responsible for such activity was found to have a stimulatory effect on the elongation factor 1 (EF-1). It copurifies with EF-1L, the lighter form of EF-1, through Sephadex G-200, DEAE-cellulose, hydroxyapatite and poly(U)-Sepharose 4B column chromatography. The two factors could be separated only through a heating step which destroyed EF-1 activity whilst leaving most of the poly(U)-binding activity unaltered.     

Structural and functional characteristics of two novel members of pathogensis-related multigene family of class 10 from yellow lupine+

PR-10 proteins (pathogensis-related), ubiquitous within the plant kingdom, are usually encoded by multigene families. To date we have identified 10 homologous pr-10 genes in a yellow lupine cDNA library. Here, the structure and expression of two newly identified yellow lupine pr-10 genes (LlYpr10-2b and LlYpr10-2f) are presented. Many potential regulatory sites were found in both gene promoters including common ones as well as those unique for each gene. However, promoter deletion analysis in transgenic tobacco plants revealed similar patterns of reporter gene (gus) expression. Shortened fragments of both gene promoters studied caused high GUS activity in leaves (along vascular bundles), stamen stigma, anthers and pollen grains. When conjugated with longer LlYpr-10.2 promoter fragments, GUS was additionally present in petal edges. Only a long fragment of the LlYpr10-2b gene promoter caused GUS expression in the stem. In yellow lupine the pr-10.2 genes are present in all studied organs, but their level of expression depends on the stage of development and is affected by wounding, oxidative stress and salicylic acid treatment. Silencing of the Llpr-10.2b gene in 4-week-old yellow lupine plants did not lead to any visible symptoms, which suggests that the function of the silenced gene is supplemented by its close homologues, still present in the studied plants.     

In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme.

A method for the solubilization of membrane-bound Cowpea mosaic virus RNA replicase has been developed by bypassing the use of detergents. Solubilization has been achieved by washing the 31,000 x g-pellet containing the bound replicase with a Mg2+-deficient buffer. This procedure had several advantages as compared to treatments with nonionic or ionic detergents: (i) the solubilized enzyme was stable at 4 C, (ii) more than 80% of the replicase could be solubilized without loss of total enzyme activity, (iii) the replicase was rather selectively released resulting in a two- to threefold increase in specific activity per se, and (iv) most of the green color from chloroplast fragments present in the crude replicase fraction remained membrane bound resulting in only slightly colored preparations of solubilized enzyme. The solubilized replicase has been further purified by DEAE-Bio Gel column chromatography. RNA synthesis directed by the DEAE-purified enzyme was template dependent and proceeded at a linear rate for at least 9 h.     

Changes in the activity and isozyme patterns of malate dehydrogenase in root nodules of yellow lupine

The malate dehydrogenase present in the cytoplasmic fraction of plant origin and bacteroids from yellow lupine root nodules was investigated. The plant enzyme was 14 times more active in nodules than in roots and it contained 6 molecular forms in nodules compared with 3 forms detected in roots. The highest malate dehydrogenase activity in plant fraction and bacteroids was noted in 50-day old plants. Changes in the isoenzymatic patterns of malate dehydrogenase in plant fraction and bacteroids accompanying ageing of the lupine root nodules were observed. Possible physiological role of malate pathway in metabolism of lupine root nodules is discussed.     

Distribution of elongation factors EF-1 and EF-2 among components of rabbit reticulocyte lysate

The distribution of activity of the elongation factors EF-1 and EF-2 among the components of rabbit reticulocyte lysate separated by sucrose density gradient centrifugation was studied. At low ionic strength (0.01 M KCl) about 30% of the EF-1 activity was found in polyribosomes. At moderate ionic strength (0.1 M KCl) the EF-1 activity was absent in the polyribosomes. An addition of RNA excess to the lysate prior to centrifugation at low ionic strength resulted in elimination of the EF-1 activity from the polyribosomes. This indicates that EF-1 is reversibly bound to the polyribosomes and that EF-1 may be retained on them due to interaction with RNA of polysomes mediated by its RNA-binding site. After dissociation of polyribosomes containing EF-1 in the presence of EDTA and subsequent fractionation of the dissociation products at low ionic strength (0.01 M KCl) the EF-1 activity was revealed in the ribosomal subparticles (predominantly in 60S). At 0.1 M KCl EF-1 mainly sedimented in the zone of distribution of polyribosomal informosomes. The elongation factor EF-2 was not revealed in polyribosomes during lysate centrifugation even at low ionic strength which corresponds to its lower affinity for RNA.     

Purification of various forms of elongation factor 1 from rabbit reticulocytes

Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (α, β, γ, and δ). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1α, EF-βδ, EF-1βγδ) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1α is a single polypeptide of M_r 52,000, and has an isoelectric point of 9.1. EF-1βδ and EF-1βγδ are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1β, 5.5 for EF-1γ, and 4.8 for EF-1δ. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1α in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1βγδ is slightly more active than EF-1βδ. The similarity of the amino acid composition of EF-1γ and EF-1δ and the molar ratio of α:β:γ:δ in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1δ is probably a breakdown product of EF-1γ, and that the native form of EF-1H probably contains only the α, β, and γ subunits.     

Purification of Euglena EF-1L: A cytoplasmic factor that also functions on procaryotic and organellar ribosomes

The low-molecular-weight form of the cytoplasmic protein synthesis elongation factor-1 (EF-1_L) from Euglena gracilis has been purified extensively from whole-cell extracts. A four-step purification procedure has been developed which results in a 45-fold enrichment in EF-1_L with 10% recovery of the total EF-1 activity present in the post-ribosomal supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the EF-1_L is greater than 90% pure. The purified factor is composed of a single subunit of molecular weight 56,000 as determined by gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. Unlike EF-1s purified to date from other organisms, Euglena EF-1_L catalyzes polymerization on Escherichia coli and Euglena chloroplast ribosomes, as well as on wheat germ ribosomes. The activity of this factor on 70 S ribosomes is about 5% that observed on eucaryotic 80 S ribosomes. This level of catalytic activity is sufficient to obscure the activity of chloroplast EF-Tu and mitochondrial EF-Tu in whole-cell extracts of Euglena. The activity of EF-1_L as measured on either wheat germ or E. coli ribosomes is unstable in the absence of glycerol, is inhibited only slightly by 20 mm, N-ethylmaleimide, is not stimulated by E. coli EF-Ts, and is not inhibited by the antibiotic kirromycin. The relative affinity of EF-1_L for guanine nucleotides was also measured and it was observed that its affinity for GTP is approximately six- to eightfold greater than that for GDP.     

Occurrence of four subunits in high molecular weight forms of polypeptide chain elongation factor 1 from wheat embryo

In contrast to high molecular weight forms of elongation factor 1 (EF-1H) from animal sources which contain three subunits, EF-1a, EF-1b, and EF-1c, EF-1H from wheat embryo consisted of four subunits, EF-1a, EF-1b, EF-1b', and EF-1c, in an equimolar ratio. The molecular weights of EF-1a, EF-1b, EF-1b', and EF-1c from wheat embryo were 52,000, 29,000, 28,000, and 48,000, respectively. In the animal system, EF-1a and EF-1b correspond functionally to EF-Tu and EF-Ts, respectively. In the wheat system, however, both EF-1b and EF-1b' had the EF-Ts-like activity to stimulate EF-1a-dependent binding of aminoacyl-tRNA to ribosomes. EF-1b and EF-1b' from wheat embryo gave 21 and 20 tryptic peptides, respectively. Twenty peptides were common.     

Immunodetection of RNA-dependent RNA polymerase from turnip yellow luteovirus using monoclonal antibodies

Monoclonal antibodies (MAs) to the RNA-dependent RNA polymerase from turnip yellow luteovirus (TYV) were prepared using a recombinant protein as immunogen and were shown to be directed to C-terminal part of the viral replicase. These MAs were found to interact with a 70-kDa protein found in extracts from TYV-infected plants. Our result is the first successful attempt at detecting the RNA-dependent RNA polymerase of a luteovirus in infected plant extracts. We also found that the protein is not processed further and its accumulation and content in the infected plant obey a definite dynamics during the infection. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Evidence for activities of rabbit reticulocyte elongation factor 1 analogous to bacterial factors EF-Ts and EF-Tu

Preparations have been obtained from rabbit reticulocyte elongation factor 1 (EF-1) that exhibit activities analogous to the heat stable and heat labile factors, EF-Ts and EF-Tu, of $\text{Escherichia coli}$. The heat stable fraction, prepared by heating EF-1 in the presence of GTP, has virtually no activity in poly (U)-directed polyphenylalanine synthesis. The fraction exhibiting activity similar to bacterial EF-Tu is obtained by the interaction of EF-1 with GTP and phenylalanyl-tRNA followed by passage of the solution through a nitrocellulose filter. The filtrate, which alone has low activity in polyphenylalanine synthesis, when combined with the heat stable fraction gives high activity suggesting that the heat stable preparation catalyzes recycling of the filtrate component.     

Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3'' end.

The genome of brome mosaic virus (BMV) is comprised of three (+) strand RNAs, each containing a similar, highly structured, 200 base long sequence at its 3' end. A 134 base subset of this sequence contains signals directing interaction of the viral RNA with BMV RNA replicase, ATP,CTP:tRNA nucleotidyl transferase and aminoacyl tRNA synthetase. A series of mutants containing deletions within this region, previously constructed and tested in vitro for the effect on replication and aminoacylation activities, has now been assayed in vitro for adenylation function and in vivo for ability to replicate in isolated protoplasts and whole plants. These tests indicate that features of viral RNA recognized by BMV replicase overlap those directing adenylation, but are distinct from those directing aminoacylation. Consequently, the lethality of a deletion preferentially inhibiting aminoacylation suggests that this function may have an essential role contributing to viral replication in vivo. An RNA3 mutant bearing a 20-base deletion yielding normal levels of aminoacylation and enhanced levels of replicase template activity and adenylation in vitro was able to replicate in protoplasts and plants; however, its accumulation in protoplasts was reduced relative to wild-type. This suggests that additional functions affecting the replication and accumulation of viral RNA reside in the conserved 3' sequence.     

Biological activities of homologous loop regions in the laminin alpha chain G domains

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.