Crystal Structure of the Intact Archaeal Translation Initiation Factor 2 Demonstrates Very High Conformational Flexibility in the α- and β-Subunits

Stolboushkina, Elena; Nikonov, S.V.; Nikulin, Alexei; Bläsi, Udo; Manstein, Dietmar J.; Fedorov, Roman; Garber, Maria; Nikonov, Oleg

doi:10.1016/j.jmb.2008.07.039

Cited by 55 publications

(62 citation statements)

References 30 publications

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“…Importantly, the growth defect associated with the corresponding mutations in yeast eIF2␥ were suppressed by overexpression of eIF2␣ (6). The binding configuration of aIF2␣ to aIF2␥ is the same in the aIF2␣␥ heterodimer and the aIF2␣␤␥ complex, consistent with the notion that aIF2␣ and aIF2␤ bind independently to aIF2␥ (3,9,10). cant insights into the mechanism of Met-tRNA i binding by eIF2 have been obtained.…”

Section: Eif2supporting

confidence: 73%

Molecular View of 43 S Complex Formation and Start Site Selection in Eukaryotic Translation Initiation

Lorsch

Dever

2010

Journal of Biological Chemistry

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A central step to high fidelity protein synthesis is selection of the proper start codon. Recent structural, biochemical, and genetic analyses have provided molecular insights into the coordinated activities of the initiation factors in start codon selection. A molecular model is emerging in which start codon recognition is linked to dynamic reorganization of factors on the ribosome and structural changes in the ribosome itself. Overview of the Eukaryotic Translation Initiation PathwayAssembly of an 80 S ribosome at the start codon of an mRNA is facilitated by translation initiation factors that function in a stepwise manner, rearranging both interfactor and factor-ribosome contacts at each step. In this review, we will highlight recent advances in our understanding of the structure-function properties of the initiation factors that function on the ribosome to promote assembly of the 43 S preinitiation complex and govern start site selection. Translation initiation ( Fig. 1) (reviewed in Ref. 1) begins with formation of TC 3 between initiator Met-tRNA i and the GTP-bound form of eIF2. The TC then associates with the small (40 S) ribosomal subunit. Binding of eIF1 and eIF1A alters the conformation of the 40 S subunit and promotes TC loading, which is also aided by eIF3. In a reaction facilitated by the eIF4 family of factors as well as by eIF3, the 43 S PIC (40 S ϩ eIF1, eIF1A, TC, eIF3) binds to an mRNA near the 5Ј cap and scans in a 3Ј direction in search of a start codon. Upon start codon recognition, eIF2 completes hydrolysis of its bound GTP in a reaction promoted by eIF5. Base pairing between the start codon on the mRNA and the anticodon loop of Met-tRNA i in the 43 S complex triggers eIF1 release from its ribosomal binding site and dissociation of P i from eIF2 to form eIF2⅐GDP, which is now unstably associated with the 40 S subunit. In a second GTP-dependent reaction, the factor eIF5B promotes joining of the large (60 S) ribosomal subunit to the 43 S complex. Hydrolysis of GTP by eIF5B following subunit joining enables eIF5B and eIF1A to dissociate from the 80 S initiation complex, leaving Met-tRNA i in the P site base paired to the start codon. The ribosome is now poised to enter the elongation phase of protein synthesis. eIF2Although the structure of the eIF2 complex, consisting of ␣, ␤, and ␥ subunits, has not yet been determined, structural studies of individual subunits as well as of the corresponding archaeal factor aIF2, in conjunction with in vivo and in vitro analyses, have recently shed light on the structure-function properties of the factor. The eIF2␣ subunit domain structure is conserved between eukaryotes and Archaea ( Fig. 2) (2, 3); however, an N-terminal extension makes the eukaryotic ␤ subunit twice the length of the archaeal protein. This extension contains three lysine-rich segments (K-boxes) consisting of 6 -8 consecutive lysine residues (Fig. 2A). The K-boxes in eIF2␤ mediate the binding of eIF2 to both its GAP, eIF5, and the catalytic subunit of its guanine nucleotide exchange facto...

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Section: Eif2supporting

confidence: 73%

Molecular View of 43 S Complex Formation and Start Site Selection in Eukaryotic Translation Initiation

Lorsch

Dever

2010

Journal of Biological Chemistry

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“…11C]), where they could act directly to inhibit GTP hydrolysis. Interestingly, the structure of heterotrimeric aIF2 from a different archaeon showed yet another orientation of the ZBD and ␣/␤ domains of aIF2␤ relative to aIF2␥, although here also the ZBD interacts with the G domain (203).…”

Section: Substitutions In Eif2␤ Increase Uug Initiation By Increasingmentioning

confidence: 78%

“…Substitutions of the corresponding residues in yeast 18S rRNA are lethal or confer an Slg Ϫ phenotype and in most cases also evoke a dominant Gcd G domain. All known structures of apo-, GDP-or GDPNPbound aIF2␥, in contrast, display a close packing of domain II with the G domain, and the switch regions do not exhibit marked conformational differences (186,203). Nevertheless, in the structure of an aIF2␣/␥-GDPNP heterodimer, the switch regions are apparently closer to the positions that they occupy in the EF-Tu/GDPNP/Phe-tRNA Phe complex than in previous aIF2␥ structures, prompting Schmitt et al to propose a model of Met-tRNA i Met docking to aIF2␥ (Fig.…”

Section: Structural Determinants Of Stringent Aug Selection In Subunimentioning

confidence: 99%

“…Considering that an equilibrium between GTP and GDP plus P i exists in the scanning PIC before AUG recognition (5), the competition for binding the G domain could be highly dynamic and might be shifted in favor of eIF5 by Sui Ϫ substitutions in eIF2␤. eIF1 also shows structural similarity to the ␣/␤ domains in aIF2␤ and eIF5, leading to the suggestion that eIF1's ␣/␤ domain might compete with that in eIF2␤ for binding to the G domain (41); however, this now seems less likely, considering that the ZBD, and not the ␣/␤ domain, of aIF2␤ contacts the G domain in recent crystal structures of heterotrimeric aIF2 (203,224). As discussed above, the ␣/␤ domains of eIF5 and eIF1 might compete for a binding site on the 40S platform in a way that regulates eIF1 dissociation upon AUG recognition (Fig.…”

Section: Vol 75 2011 Scanning and Aug Selection In Eukaryotes 453mentioning

confidence: 99%

“…The N-terminal domain (domain I) of aIF2␣ contains a ␤-barrel, with nonspecific RNA-binding activity (225), and interacts with the central, helical domain (domain II) through a hydrophobic core. In the heterotrimeric aIF2 structure, domains I and II of aIF2␣ make no contacts with other aIF2 subunits, and their orientation relative to domain III is highly variable among published structures (203). In vitro assays suggest that the association with aIF2␣ domain III increases the affinity of aIF2␥ for Met-tRNA i Met by orders of magnitude, whereas the ␤ subunit makes only a small contribution to tRNA binding by aIF2␥ (223,225).…”

Section: Roles Of Eif2␣ In Initiator Trna Binding and Recognition Of mentioning

confidence: 99%

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Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

Hinnebusch

2011

Microbiol Mol Biol Rev

364

415

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SUMMARY The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNA i Met ) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNA i Met bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNA i Met more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.

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Ribosomal Protein S5e is Implicated in Translation Initiation through its Interaction with the N‐Terminal Domain of Initiation Factor eIF2α

Sharifulin¹,

Babaylova²,

Kossinova³

et al. 2013

ChemBioChem

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A key step of translation initiation in eukaryotes is formation of the 48S preinitiation complex (PIC) containing the 40S ribosome, a set of eukaryotic initiation factors (eIFs), mRNA, and initiator Met-tRNA interacting with mRNA start codon; however, the PIC structure remains substantially unknown. Here, we apply formaldehyde-induced protein-protein crosslinks to identify contacts between ribosomal protein S5e (rpS5e, "e" stands for "eukaryotic") and eIFs within the mammalian PIC, assembled on either model canonical or IRES-containing mRNA. Using immunoblotting and mass spectrometry, we show that with both types of mRNA, rpS5e crosslinks to eIF2α. Comparative analysis of peptides resulting from trypsinolysis of the crosslinked proteins before and after crosslink reversal reveals crosslinked peptides in the N-terminal parts of rpS5e and eIF2α. Application of these data to a model PIC structure obtained with the use of available structures indicates that eIF2α undergoes major conformation rearrangements to enable contacts of the factor with rpS5e. These contacts are suggested to maintain the correct positioning of eIF2α relative to other PIC components; this could be essential for start-codon selection by the PIC.

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Crystal Structure of the Intact Archaeal Translation Initiation Factor 2 Demonstrates Very High Conformational Flexibility in the α- and β-Subunits

Cited by 55 publications

References 30 publications

Molecular View of 43 S Complex Formation and Start Site Selection in Eukaryotic Translation Initiation

Molecular View of 43 S Complex Formation and Start Site Selection in Eukaryotic Translation Initiation

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

Ribosomal Protein S5e is Implicated in Translation Initiation through its Interaction with the N‐Terminal Domain of Initiation Factor eIF2α

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