SUMMARY Transcript-selective translational regulation of epithelial-mesenchymal transition (EMT) by transforming growth factor-β (TGFβ) is directed by the hnRNP E1-containing TGFβ-activated-translational (BAT) mRNP complex. Herein, eukaryotic elongation factor-1 A1 (eEF1A1) is identified as an integral component of the BAT complex. Translational silencing of Dab2 and ILEI, two EMT-transcripts, is mediated by binding of hnRNP E1 and eEF1A1 to their 3′-UTR BAT element, whereby hnRNP E1 stalls translational elongation by inhibiting the release of eEF1A1 from the ribosomal A site. TGFβ-mediated hnRNP E1 phosphorylation, through Akt2, disrupts the BAT complex, thereby restoring translation of target EMT-transcripts. Attenuation of hnRNP E1 expression in two non-invasive breast epithelial cells (NMuMG and MCF-7) induced not only EMT, but also enabled cells to form metastatic lesions in vivo. Thus, translational regulation by TGFβ, at the elongation stage, represents a critical checkpoint coordinating the expression of EMT-transcripts required during development and in tumorigenesis and metastatic progression.

Eukaryotic elongation factor 1 A (eEF1A, formerly elongation factor-1 alpha) is an important component of the protein synthesis apparatus. Here we report the isolation and characterization of the cDNA sequence encoding rabbit eEF1A-2, an isoform of eEF1A, as well as a structural and functional comparison of the two rabbit isoforms. Northern analysis of the expression pattern of eEF1A-2 showed that this isoform is expressed in skeletal muscle, heart, brain and aorta, while transcripts are not detected in liver, kidney, spleen and lung. In contrast, the previously characterized eEF1A-1 isoform is expressed in all tissues examined except skeletal muscle. We have recently purified eEF1A-2 from rabbit skeletal muscle. By partial amino acid sequencing and determination of the post-translational modifications of eEF1A-2 we found that both of the glycerylphosphorylethanolamine modifications observed in eEF1A-1 appear to be present in eEF1A-2. However, two of the residues found dimethylated in eEF1A-1 appeared to be trimethylated in eEF1A-2. A comparison of the enzymatic activity showed that eEF1A-1 and eEF1A-2 have indistinguishable activity in an in vitro translation system. In contrast, the GDP dissociation rate constant is approximately 7 times higher for eEF1A-1 than for eEF1A-2. The nucleotide preference ratio (GDP/GTP) for eEF1A-1 was 0.82, while the preference ratio for eEF1A-2 was 1.50.

P-type ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest a transport mechanism defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have employed single-molecule fluorescence resonance energy transfer (smFRET) to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. Using the smFRET approach we identify key intermediates with no known crystal structures, and our findings delineate reversible and an essentially irreversible step in the transport process wherein Ca2+ efflux by LMCA1 is rate limited by phosphoenzyme formation and resolved by ADP and Ca2+ release leading to an open E2P state.

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qβ is composed of the viral catalytic β-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the β-subunit and the two host proteins to 2.5-Å resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the β-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the β-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.protein biosynthesis | virus

In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1B alpha, eEF1B eta and eEF1B gamma. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1B beta and eEF1B gamma subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein-protein interactions. The following complexes were observed: eEF1A1:eEF1B alpha, eEF1A1:eEF1B beta, eEF1B beta:eEF1B beta, eEF1B alpha:eEF1B gamma, eEF1B beta:eEF1B gamma and eEF1B alpha:eEF1B gamma:eEF1B beta, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1B alpha or eEF1B beta held together by eEF1B gamma. These units can dimerize via eEF1B beta. Our model is compared with other models, and structural as well as functional aspects of the model are discussed.