The eEF1 family of mammalian translation elongation factors.

The eEF1 family of mammalian translation elongation factors is comprised of the two variants of eEF1A (eEF1A1 and eEF1A2), and the eEF1B complex. The latter consists of eEF1Bα, eEF1Bß, and eEF1Bγ subunits. The two eEF1A variants have similar translation activity but may differ with respect to their secondary, "moonlighting" functions. This variability is underlined by the difference in the spatial organization of eEF1A1 and eEF1A2, and also possibly by the differences in their post-translational modifications. Here, we review the data on the spatial organization and post-translation modifications of eEF1A1 and eEF1A2, and provide examples of their involvement in various processes in addition to translation. We also describe the structural models of eEF1B subunits, their organization in the subcomplexes, and the trimeric model of the entire eEF1B complex. We discuss the functional consequences of such an assembly into a complex as well as the involvement of individual subunits in non-translational processes.

[Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)].

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.

Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian translation elongation factor eEF1A2.

Translation elongation factor eEF1A2 was purified to homogeneity from rabbit muscle by two consecutive ion-exchange column-chromatography steps and this mammalian eEF1A2 was successfully crystallized for the first time. Protein crystals obtained using ammonium sulfate as precipitant diffracted to 2.5 Å resolution and belonged to space group P6(1)22 or P6(3)22 (unit-cell parameters a = b = 135.4, c = 304.6 Å). A complete native data set was collected to 2.7 Å resolution.

Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H.

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.

Evidence for the formation of an unusual ternary complex of rabbit liver EF-1alpha with GDP and deacylated tRNA.

Eukaryotic translation elongation factor 1alpha is known to interact in GTP-bound form with aminoacyl-tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF-1alpha*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18-20), has been detected by four independent methods. [EF-1alpha*GDP]-interacting sites are located in the acceptor stem, TpsiC stem and TpsiC loop of tRNA(Phe) and tRNA(Leu) molecules. Both tRNA and EF-1alpha are found to undergo certain conformational changes during their interaction. The ability of EF-1alpha to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl-tRNA channeling in higher eukaryotic cells.

A fast and effective method for purification of elongation factor 1 alpha from rabbit liver.

Fast, efficient and gentle method of elongation factor 1 alpha (EF-1 alpha) purification from rabbit liver has been developed. Isolation procedure consists of three steps: gel-filtration of postmithohondrial supernatant on Sephacryl S-400, anion(DEAE-Cellulose)- and cation(SP-Sepharose)- exchange chromatographies. The procedure takes two-three days. Minimal number of the purification steps and the only one buffer usage during entire purification provide the high speed and efficiency of the method. Purity of EF-1 alpha preparation obtained was more than 90% as judged from DS-Na polyacrylamide gel electrophoresis. EF-1 alpha was demonstrated to be highly active in three different assays: the [3H]GDP-binding, the stimulation of [14C]phenylalanyl-tRNA binding to poly(U)-programmed 80S ribosomes and the stimulation of poly[14C]phenylalanine synthesis on poly(U)-programmed 80S ribosomes in the presence of EF-2. Since EF-1 alpha may exist in a cell as either GDP- or GTP-bound form the nature of nucleotide bound was studied using high pressure liquid chromatography (HPLC) analysis. Each molecule of EF-1 alpha appears to contain one molecule of GDP.

Rabbit translation elongation factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase.

Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964-968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation elongation factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis.