Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Gilbert, Robert J.C.; Gordiyenko, Yulya; Haar, Tobias von der; Sonnen, Andreas F.‐P.; Hofmann, Gregor; Nardelli, Maria; Stuart, David I.; McCarthy, John E.G.

doi:10.1073/pnas.0606880104

Cited by 22 publications

(23 citation statements)

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“…This conformation would likely promote mRNA binding and allow the ribosome to move more easily across the mRNA. A similar result was achieved in a cryo-EM reconstruction of the 40 S subunit bound to eIF1A and the multifactor complex (MFC), which is composed of eIF1, eIF3, eIF5, and TC (27). These results are consistent with earlier hydroxyl radical cleavage studies that suggested that structural changes occur in the mRNA binding region of the ribosome upon association of eIF1 (28).…”

Section: Searching For the Start Codonsupporting

confidence: 80%

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Mitchell

Lorsch

2008

Journal of Biological Chemistry

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Start codon selection is a key step in translation initiation as it sets the reading frame for decoding. Two eukaryotic initiation factors, eIF1 and eIF1A, are key actors in this process. Recent work has elucidated many details of the mechanisms these factors use to control start site selection. eIF1 prevents the irreversible GTP hydrolysis that commits the ribosome to initiation at a particular codon. eIF1A both promotes and inhibits commitment through the competing influences of its two unstructured termini. Both factors perform their tasks through a variety of interactions with other components of the initiation machinery, in many cases mediated by the unstructured regions of the two proteins.Translation initiation begins the final major step in gene expression and is a highly regulated process. Initiation entails the formation of a functional ribosomal complex (80 S in eukaryotes) with an initiator methionyl-tRNA (Met-tRNA i ) bound in the P-site, its anticodon base-paired to the start codon of an mRNA, poised to begin peptide synthesis. Formation of the initiation complex is promoted and regulated by a number of non-ribosomal proteins called initiation factors (IFs, 2 or for eukaryotes, eIFs).Many details of eukaryotic translation initiation have been elucidated over the last three decades using genetic, biochemical, and structural techniques. As this review focuses on the roles of eIF1 and eIF1A in start site selection, only a brief overview of the entire process will be given ( Fig. 1). For a more complete description, see one of several recent comprehensive reviews (1-3). Met-tRNA i is delivered to the P-site of the small (40 S) ribosomal subunit in the form of a ternary complex (TC) including GTP and the trimeric GTPase eIF2, forming the 43 S pre-initiation complex (PIC) in a process promoted by eIF1, eIF1A, and eIF3. The 43 S PIC then binds the 5Ј-end of an mRNA with the help of eIF3, eIF4A, eIF4B, and eIF4F, creating the 48 S PIC. The mRNA is thought to be circularized through the interactions of PABP with the 3Ј-poly(A) tail of the mRNA and the eIF4F 5Ј-cap-binding complex. The complex is then believed to scan along the mRNA in search of the start codon in an ATP-dependent process. During this search, eIF2 partially hydrolyzes its bound GTP with the help of the GTPase-activating factor eIF5. Prior to start codon recognition, the resultant phosphate ion is not released, producing GTP-and GDP⅐P ibound states of the factor, possibly in equilibrium (4). Upon identification of the start codon, the phosphate is released, making GTP hydrolysis irreversible and committing the complex to proceeding with initiation at that site on the mRNA. After eIF2⅐GDP release, the 60 S subunit can join the 40 S subunit with the help of a second GTPase, eIF5B. When eIF5B⅐GDP dissociates after subunit joining, the initiation complex is complete and ready to begin the elongation phase of translation.In eukaryotic cells, at least twelve factors, composed of over two dozen polypeptides, are required for initiation complex forma...

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Section: Searching For the Start Codonsupporting

confidence: 80%

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Mitchell

Lorsch

2008

Journal of Biological Chemistry

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“…Rotation of the head was also observed in cryo-EM reconstructions of eukaryotic 40S subunits on binding to internal ribosome entry sites (IRESs) of viral mRNAs (Spahn et al 2001b(Spahn et al , 2004b or to the multifactor complex of yeast, comprised of eIF3, eIF1, eIF5 and TC, and eIF1A (Gilbert et al 2007). It is intriguing that the C934T Gcd − mutation we obtained alters the unpaired residue at the base of h28 that serves as the pivot point for head rotation in 70S complexes (Schuwirth et al 2005).…”

mentioning

confidence: 85%

Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNA^Met and AUG selection

Dong¹,

Nanda

Rahman

et al. 2008

Genes Dev.

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High-resolution structures of bacterial 70S ribosomes have provided atomic details about mRNA and tRNA binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (PICs). We identified residues in yeast 18S rRNA critical in vivo for recruiting methionyl tRNA i Met to 40S subunits during initiation by isolating mutations that derepress GCN4 mRNA translation. Several such Gcd − mutations alter the A928:U1389 base pair in helix 28 (h28) and allow PICs to scan through the start codons of upstream ORFs that normally repress GCN4 translation. The A928U substitution also impairs TC binding to PICs in a reconstituted system in vitro. Mutation of the bulge G926 in h28 and certain other residues corresponding to direct contacts with the P-site codon or tRNA in bacterial 70S complexes confer Gcd In protein synthesis, each nucleotide triplet in mRNA is decoded by the cognate aminoacyl-tRNA in the aminoacyl (A) site of the ribosome, while peptidyl-tRNA containing the growing polypeptide chain base-pairs with the preceding triplet in the peptidyl (P) site. During initiation, in contrast, the AUG start codon is decoded in the P-site by Met-tRNA i Met. In bacteria, the Shine-Dalgarno sequence in mRNA base-pairs with 16S rRNA to help position the AUG start codon in the P-site. Accurate start codon selection also depends on translation initiation factors IF1, IF2, and IF3, of which IF1 and IF3 stimulate binding of Met-tRNA i Met to the preinitiation complex (PIC) in preference to elongator tRNA species (Antoun et al. 2006). The ribosomal determinants of tRNA binding to the P-site in bacteria are being illuminated by high-resolution crystal structures of 70S ribosomes bound to mRNA with cognate tRNA in the P-site. On the small (30S) subunit, 11 residues of 16S rRNA directly contact the P-site codon, the P-tRNA anticodon, or the P-tRNA anticodon stem-loop (ASL) (Berk et al. 2006;Korostelev et al. 2006;Selmer et al. 2006). In particular, G1338 and A1339 interact with G-C base pairs in the ASL of tRNA i Met , and functional studies have implicated these 16S residues in promoting recruitment of initiator versus elongator tRNA and accurate selection of AUG as start codon (Lancaster and Noller 2005;Qin et al. 2007).Initiation in eukaryotes is more complex, with 13 different initiation factors (eIFs) participating in a multistep pathway. The eIF2, in its GTP-bound state, forms a ternary complex (TC) with Met-tRNA i Met and binds to the 40S subunit to form the 43S PIC in a manner stimulated by eIF1, eIF1A, eIF5, and eIF3. The 43S PIC then binds to the 5Ј end of mRNA, preactivated by eIF4F bound to the cap. The AUG codon is selected as the 43S PIC scans the leader, with the anticodon of Met-tRNA i Met inspecting successive triplets, presumably, as they enter the P-site. eIF1 and eIF1A promote the scanning process at least partly by stabilizing an open conformation of the 40S subunit when non-AUGs occupy the P-site. The GTP

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“…This technological platform is beginning to enable investigators to fit genetic and biochemical knowledge into a structural context. For example, whereas there is a wealth of genetic and biochemical information pertaining to translation initiation in yeast, cryo-EM studies are revealing specific structural rearrangements in the 40 S subunit consequent to binding and release of specific initiation factors (14,15). Similarly, cryo-EM methods are illuminating the details of the interactions between the ribosome and the signal recognition particle (reviewed in Ref.…”

Section: Structural Biologymentioning

confidence: 99%

The Eukaryotic Ribosome: Current Status and Challenges

Dinman

2009

Journal of Biological Chemistry

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Despite having been identified first, their greater degree of complexity has resulted in our understanding of eukaryotic ribosomes lagging behind that of their bacterial and archaeal counterparts. A much more complicated biogenesis program results in ribosomes that are structurally, biochemically, and functionally more complex. However, recent advances in molecular genetics and structural biology are helping to reveal the intricacies of the eukaryotic ribosome and to address many longstanding questions regarding its many roles in the regulation of gene expression.Since its initial discovery using differential ultracentrifugation of rat liver homogenates (reviewed in Ref. 1), the ribosome has remained a foundational platform upon which our understanding of the relationship between structure and function at the molecular level has been built. There is a rich history of biochemistry and genetics of eukaryotic ribosomes, including the discovery in the 1950s that they 32 are the site of protein synthesis, the elucidation of the function of the nucleolus, and even the discovery of the first eukaryotic RNA polymerase (reviewed in Ref. 2). Whereas early studies using mammalian ribosomes defined the "integral requirements" for protein synthesis, a switch to bacterial ribosomes in the 1960s facilitated the identification of the "minimal requirements" for the translational machinery, giving rise to a "golden age" of translation. In particular, the greater degree of structural and functional complexity makes eukaryotic ribosomes more challenging to work with than their bacterial and archaeal counterparts. For example, whereas bacterial translation initiation requires only a small set of trans-acting factors and is facilitated by the ShineDalgarno sequence, this process in eukaryotes requires a multifactorial complex of trans-acting factors that is almost as massive as the ribosome itself (reviewed in Ref. 3). Here, some of the current topics and challenges in the study of the eukaryotic ribosome are reviewed.

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Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Cited by 22 publications

References 35 publications

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNA^Met and AUG selection

The Eukaryotic Ribosome: Current Status and Challenges

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Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Cited by 22 publications

References 35 publications

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNAMet and AUG selection

The Eukaryotic Ribosome: Current Status and Challenges

Contact Info

Product

Resources

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Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNA^Met and AUG selection