In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I.

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The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met-tRNA i Met ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui ؊ phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui ؊ phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui ؊ phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.Translation initiation is a multistep process culminating in formation of the 80S initiation complex containing methionyl initiator tRNA (Met-tRNA i Met ) base paired with the AUG start codon in the P site of the ribosome. A large number of soluble eukaryotic translation initiation factors (eIFs) have been identified that stimulate the partial reactions of this process (reviewed in reference 12 and 13). A critical step early in the pathway is the binding of Met-tRNA iMet to the 40S ribosomal subunit in a ternary complex (TC) comprised of MettRNA i Met , GTP, and eIF2. The recruitment of TC to 40S subunits is promoted in vitro by eIF1, eIF1A, and the eIF3 complex. The 43S preinitiation complex thus formed interacts with mRNA in a manner stimulated by eIF4F (eIF4A-eIF4E-eIF4G), poly(A)-binding protein, and eIF3, and the 43S complex scans the mRNA until the Met-tRNA i Met base pairs with an AUG triplet. AUG recognition triggers GTP hydrolysis by eIF2 in a reaction stimulated by eIF5, and the eIF2-GDP and other eIFs are ejected from the ribosome. The eIF1, eIF1A, and eIF4G have been implicated in the scanning process in vitro (23, 24). In the final reaction, eIF5B bound to GTP promotes joining of the 60S subunit with the 40S-MettRNA i Met -mRNA complex to produce the 80S initiation complex (15,25). To begin a new round of initiation, the ejected eIF2-GDP complex must be recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B (13).From extensive biochemical analysis of the mammalian initiation factors, it was proposed that eIF3 binds to the 40S ribosome independently of other factors and promotes the recruitment of TC and mRNA in a manner stimulated by eIF1 ...

Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5 of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5 enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.[Keywords: Translation initiation; reinitiation; eIF3; 40S ribosomal subunit; GCN4; short uORF] Supplemental material is available at http://www.genesdev.org.

RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors. RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes. Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size. There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal. A mutation in the second ATPbinding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts. These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation. RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells. Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis. Most of the proteins belonging to the ATP-binding cassette (ABC)1 superfamily of proteins are membrane transporters that use ATP hydrolysis to transport solute molecules against a concentration gradient (1). Typically, they contain two ABCs and a transmembrane domain. The ABC contains a nucleotide binding domain with Walker A and B motifs and an ␣-helical domain bearing the "LSGGQ" signature motif that, along with several other conserved features, distinguishes ABC proteins from other ATPases. ABCs bind ATP as a dimer, with the LSGGQ motif of one cassette capping the ATP molecule bound to the Walker motifs of the second cassette in the dimer. This produces an "ATP sandwich" with two ATP molecules bound to two hybrid active sites formed by the dimerized cassettes. ATP hydrolysis destabilizes the dimer, and it was proposed that a cycle of dimerization driven by ATP binding and hydrolysis can perform mechanical work. For ABC transporters, this would entail opening and closing a solute channel in the membrane through conformational changes in the transmembrane domains (2-6).The yeast proteins GCN20 and YEF3 are soluble ABC proteins whose functions are connected with the binding of tRNAs to ribosomes. YEF3 (eEF3) is an essential translation elongation factor that stimulates release of deacylated tRNA from the ribosomal E-site concurrent with the recruitment of aminoacylated tRNA to the ribosomal A-site (7). GCN20 is a positive regulator of GCN2, a protein kinase that reg...

The diagnosis of brucellosis in livestock and wildlife is complex and serological results need to be carefully analyzed. The B. abortus S19 and B. melitensis Rev. 1 vaccines are the cornerstones of control programs in cattle and small ruminants, respectively. There is no vaccine available for pigs or for wildlife. In the absence of a human brucellosis vaccine, prevention of human brucellosis depends on the control of the disease in animals.

. These results suggest that eIF3 binds to the solvent side of the 40S subunit in a way that provides access to the interface side for the two eIF3 segments (NIP1-NTD and TIF32-CTD) that interact with eIF1, eIF5, and the eIF2/GTP/Met-tRNA i Met ternary complex.[Keywords: Eukaryotic translation initiation factor (eIF); multifactor complex (MFC); translational control; protein synthesis; 40S ribosome binding; TIF32/NIP1] Supplemental material is available at http://www.genesdev.org.

DEAH helicases participate in pre-messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p-ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide-binding motif. A b-hairpin from the second RecA domain is wedged between two carboxyterminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C-terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides the framework for functional and genetic analysis of all DEAH helicases.