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The signal recognition particle (SRP) is a conserved ribonucleoprotein particle that targets membrane and secreted proteins to translocation channels in membranes. In eukaryotes, the Alu domain, which comprises the 5′ and 3′ extremities of the SRP RNA bound to the SRP9/14 heterodimer, is thought to interact with the ribosome to pause translation elongation during membrane docking. We present the 3.2 Å resolution crystal structure of a chimeric Alu domain, comprising Alu RNA from the archaeon Pyrococcus horikoshii bound to the human Alu binding proteins SRP9/14. The structure reveals how intricate tertiary interactions stabilize the RNA 5′ domain structure and how an extra, archaeal-specific, terminal stem helps constrain the Alu RNA into the active closed conformation. In this conformation, highly conserved noncanonical base pairs allow unusually tight side-by-side packing of 5′ and 3′ RNA stems within the SRP9/14 RNA binding surface. The biological relevance of this structure is confirmed by showing that a reconstituted full-length chimeric archaeal-human SRP is competent to elicit elongation arrest in vitro. The structure will be useful in refining our understanding of how the SRP Alu domain interacts with the ribosome.

International audience; In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3-A resolution crystal structure of the Escherichia coli WH3/4 domains bound to mRNA with mutagenesis data reveal that the two WH motifs use the same structural elements to bind RNA. The structure together with the 2.6-A resolution structure of the WH1-4 domains from Moorella thermoacetica bound to RNA revealed that a salt bridge connecting WH2 to WH3 modules is disrupted upon mRNA binding. The results provide a structural basis for the molecular switch that may allow communication between tRNA and mRNA binding sites and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein-RNA complexes.

International audience; Elongation factor 1 mediates the elongation step of mRNA translation. The transfer of aminoacyl-tRNA to ribosomes under hydrolysis of GTP is catalyzed by a GTP binding protein, EF1Ia. A guanine-nucleotide exchange complex now referred as EF1beta gamma delta replaces GDP by GTP on EF1alpha (1). A complex, purified from Xenopus oocytes as a substrate for the meiotic and mitotic p34Cdc2 kinase, was shown to contain the guanine-nucleotide exchange activity (2). The Xenopus complex is composed of three main proteins, p30, p36 and p47. Surprisingly, microsequencing of two of its components, p36 and p30 suggested the presence of two related proteins (2). We have previously cloned and sequenced the cDNA encoding for p47 or EF1gamma (3) and p36 or EF1delta (4), we present here the molecular cloning of p30 or EF1beta. This result allows for the first time, sequence analysis of EF1beta and delta proteins, both present in the same complex.

International audience; In the amphibian Xenopus laevis, the elongation factor 1 alpha proteins (EF-1 alpha) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF-1 alpha gene (EF-1 alpha S) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF-1 alpha (conversion of EF-1 alpha-GDP to EF-1 alpha-GTP) is assured by the EF-1 beta gamma complex. We show here that in Xenopus laevis embryos, contrary to the situation for EF-1 alpha, EF-1 beta, and EF-1 gamma mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF-1 beta and EF-1 gamma genes from the zygotic genome occurs several hours after that of the somatic EF-1 alpha S gene. Therefore, during early Xenopus development the expression of these three elongation factors is not co-ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the early Xenopus embryo are discussed.

International audience; In A. thaliana the translation elongation factor EF-1 alpha is encoded by a small multigenic family of four members (A1-A4). The A1 gene promoter has been dissected and examined in a transient expression system using the GUS reporter gene. Deletion analysis has shown that several elements are involved in the activation process. One cis-acting domain, the TEF 1 box, has been accurately mapped 100 bp upstream of the transcription initiation site. This domain is the target for trans-acting factors identified in nuclear extracts prepared from A. thaliana. Homologies are found between the TEF 1 box and sequences present at the same location within the A2, A3 and A4 promoters. This observation, together with those obtained from gel retardation assays performed using DNA fragments from the A4 promoter, suggest that the activation process mediated by the TEF 1 element is conserved among the A. thaliana EF-1 alpha genes. Analysis of nearly full length cDNA clones has shown that in addition to a single intron located within the coding region, the A1 gene contains a second intron located within the 5' non coding region. Such an intron is also present within the A2, A3 and A4 genes. This 5' intervening sequence appears to be essential to obtain a maximum GUS activity driven by the A1 gene promoter.

International audience

International audience; Subsequent to their aminoacylation, tRNAs are subject to specific maturation and/or correction processes. Aminoacylated tRNAs ready for use in translation are then specifically channelled to the ribosomal A or P sites. Structural and biochemical studies have opened the way towards furthering our understanding of these routes to the ribosome, which involve a strict distinction between initiator and elongator tRNAs.