Molecular mechanisms of translation initiation in eukaryotes (original) (raw)
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Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes
The EMBO Journal, 2008
The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions þ 11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and þ 9-þ 11 and þ 8-þ 9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions À 3/ À 4 to rpS5(S7p) and h23b, À 6/ À 7 to rpS14(S11p), and À 8-À 11 to the 3 0 -terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions þ 7/ þ 8 to the central region of h28, þ 4/ þ 5 to rpS15(S19p), and À 6 and À 7/ À 10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2a (eIF2a) contacts position À 3, and now report that eIF3 interacts with positions À 8-À 17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.
Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons
Nature, 1998
The scanning model of translation initiation is a coherent description of how eukaryotic ribosomes reach the initiation codon after being recruited to the capped 5' end of messenger RNA. Five eukaryotic initiation factors (eIF 2, 3, 4A, 4B and 4F) with established functions have been assumed to be sufficient to mediate this process. Here we report that eIF1 and eIF1A are also both essential for translation initiation. In their absence, 43S ribosomal preinitiation complexes incubated with ATP, eIF4A, eIF4B and eIF4F bind exclusively to the cap-proximal region but are unable to reach the initiation codon. Individually, eIF1A enhances formation of this cap-proximal complex, and eIF1 weakly promotes formation of a 48S ribosomal complex at the initiation codon. These proteins act synergistically to mediate assembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA.
Genes & Development, 2000
Translation initiation factor 2 (eIF2) bound to GTP transfers the initiator methionyl tRNA to the 40S ribosomal subunit. The eIF5 stimulates GTP hydrolysis by the eIF2/GTP/Met-tRNAiMet ternary complex on base-pairing between Met-tRNAiMet and the start codon. The eIF2, eIF5, and eIF1 all have been implicated in stringent selection of AUG as the start codon. The eIF3 binds to the 40S ribosome and promotes recruitment of the ternary complex; however, physical contact between eIF3 and eIF2 has not been observed. We show that yeast eIF5 can bridge interaction in vitro between eIF3 and eIF2 by binding simultaneously to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIF2β, dependent on a conserved bipartite motif in the carboxyl terminus of eIF5. Additionally, the amino terminus of NIP1 can bind concurrently to eIF5 and eIF1. These findings suggest the occurrence of an eIF3/eIF1/eIF5/eIF2 multifactor complex, which was observed in cell extracts free of 40S ribosomes...
Molecular Cell, 2007
Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2GTPMet-tRNA i Met ) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.
Biochimie, 1991
m More than ten different protein factors are involved in initiation of protein synthesis in eukaryotes. For binding of initiator tRNA and mRNA to the 40S ribosomal subunit, the initiation factors elF-2 and elF-3 are particularly important. They consist of several different subunits and form stable complexes with the 40S ribosomal subunit. The location of elF-2 and elF-3 in these complexes as well as the interactions of the individual components have been analyzed by biochemical methods and electron microscopy. The results obtained are summarized in this article, and a model is derived describing the spatial arrangement of eIF-2 and elF-3 together with initiator tRNA and mRNA on the 40S subunit. Conclusions on the location of functionally importm~t sites of eukaryotic small ribosomal subunits are discussed with regard to the respective location of these sites in the prokaryotic counterpart.
Journal of Biological Chemistry, 1998
A complex of eukaryotic initiation factors (eIFs) 4A, 4E, and 4G (collectively termed eIF4F) plays a key role in recruiting mRNAs to ribosomes during translation initiation. The site of ribosomal entry onto most mRNAs is determined by interaction of the 5-terminal cap with eIF4E; eIFs 4A and 4G may facilitate ribosomal entry by modifying mRNA structure near the cap and by interacting with ribosome-associated factors. eIF4G recruits uncapped encephalomyocarditis virus (EMCV) mRNA to ribosomes without the involvement of eIF4E by binding directly to the ϳ450-nucleotide long EMCV internal ribosome entry site (IRES). We have used chemical and enzymatic probing to map the eIF4G binding site to a structural element within the J-K domain of the EMCV IRES that consists of an oligo(A) loop at the junction of three helices. The oligo(A) loop itself is not sufficient to form stable complexes with eIF4G since alteration of its structural context abolished its interaction with eIF4G. Addition of wild type or trans-dominant mutant forms of eIF4A to binary IRES⅐eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.