Characterization of Yeast Translation Initiation Factor 1A and Cloning of Its Essential Gene

Wei, Chia‐Lin; Kainuma, Mami; Hershey, John W.B.

doi:10.1074/jbc.270.39.22788

Cited by 42 publications

(32 citation statements)

References 36 publications

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“…The presence of eIF1A in the 40 S initiation complex was also shown in one of these earlier studies (9). In vivo studies in Saccharomyces cerevisiae demonstrated that the genes encoding these two small initiation factors are essential for initiation of protein synthesis and required for cell growth and viability (12)(13)(14). These observations are in accord with genetic studies in S. cerevisiae (15,16) that indicate that eIF1 (SUI 1) plays an essential role in the fidelity of start site selection.…”

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confidence: 79%

Mammalian Translation Initiation Factor eIF1 Functions with eIF1A and eIF3 in the Formation of a Stable 40 S Preinitiation Complex

Majumdar

Bandyopadhyay²,

Maitra

2003

Journal of Biological Chemistry

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We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA i . These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S⅐eIF3⅐eIF1⅐eIF1A⅐Met-tRNA i ⅐eIF2⅐GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.The initiation of translation in eukaryotic cells occurs by a sequence of partial reactions that require a number of specific proteins called eukaryotic (translation) initiation factors (eIFs).1 According to the currently accepted view of translation initiation, primarily derived from in vitro studies with purified initiation factors, an obligatory intermediate step in the overall initiation reaction is the binding of the initiator Met-tRNA i as the Met-tRNA i ⅐eIF2⅐GTP ternary complex to a 40 S ribosomal subunit containing bound initiation factor eIF3. This interaction leads to the production of the 40 S preinitiation complex (40 S⅐eIF3⅐Met-tRNA i ⅐eIF2⅐GTP). The 40 S preinitiation complex then binds to the 5Ј-capped end of mRNA and scans the mRNA in a 5Ј33Ј direction until the 40 S complex encounters the initiating AUG codon to form the 40 S initiation complex (40 S⅐eIF3⅐mRNA⅐Met-tRNA i ⅐eIF2⅐GTP). This reaction requires the participation of three other initiation factors eIF4F, eIF4A, and eIF4B. Subsequently, the 60 S ribosomal subunit joins the 40 S complex in a reaction dependent on two other factors, eIF5 and eIF5B, to form a functional 80 S initiation complex (80 S⅐mRNA⅐Met-tRNA i ) (for review, see Refs. 1-5).In addition to the initiation factors described above, the 17-kDa eIF1A and the 12-kDa eIF1 are also known to play essential roles in the overall initiation process (1-5). Earlier biochemical studies demonstrated that both eIF1 and eIF1A have a weak stimulatory effect on the binding of Met-tRNA i and mRNA to 40 S and 80 S initiation complexes in the presence of other factors (6 -11). The presence of eIF1A in the 40 S initiation complex was also shown in one of these earlier studies (9). In vivo studies in Saccharomyces cerevisiae demonstrated that the genes encoding these two small initiation factors are essential for initiation of protein synthesis an...

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confidence: 79%

Mammalian Translation Initiation Factor eIF1 Functions with eIF1A and eIF3 in the Formation of a Stable 40 S Preinitiation Complex

Majumdar

Bandyopadhyay²,

Maitra

2003

Journal of Biological Chemistry

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“…We used yeast two-hybrid assays to identify and then localize the eIF1A-interacting region in eIF5B. A series of GAL4 DNA binding domain and GAL4 activation domain fusions containing various truncated fragments of yeast eIF5B were tested for interaction with the appropriate fusions containing the full-length 153-amino-acid yeast eIF1A (43) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

Choi

Olsen

Roll-Mecak

et al. 2000

Molecular and Cellular Biology

101

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To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

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“…Cultures were grown at 30ЊC and were monitored by measuring the optical density at 600 nm (OD 600 ). For sporulation (20), cells were grown on YPD plates (containing 6% glucose) for 24 h and then were sporulated at room temperature on plates modified for strain W303 (27) containing 0.3% potassium acetate, 0.02% raffinose, and 10 g of each amino acid per ml. Tetrad dissections and DNA transformations were carried out by using standard procedures (25).…”

Section: Methodsmentioning

confidence: 99%

The 39-Kilodalton Subunit of Eukaryotic Translation Initiation Factor 3 Is Essential for the Complex’s Integrity and for Cell Viability in Saccharomyces cerevisiae

Naranda

Kainuma

Macmillan

1997

Molecular and Cellular Biology

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Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNA i and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.Initiation of protein synthesis is promoted by at least 10 proteins called initiation factors (reviewed in reference 14). The largest and most complex of these is eukaryotic translation initiation factor 3 (eIF3), a factor comprising at least eight subunits. eIF3 plays a central role in the initiation pathway in mammalian cells (1a, 26). It binds to 40S ribosomal subunits and is implicated in dissociating 80S ribosomes into 40S and 60S subunits (1a, 6). It prevents dissociation of the MettRNA i ⅐ eIF2 ⅐ GTP ternary complex caused by addition of RNA (7) and stabilizes ternary complex binding to 40S ribosomal subunits (1a). eIF3 is required for mRNA binding to 40S and 80S ribosomes (1a, 26), in part by binding the eIF4G subunit of the cap-binding complex, eIF4F (12, 13). Therefore, knowledge of the structure and function of eIF3 is essential for understanding the mechanism of the initiation phase of protein synthesis.To better elucidate the function of eIF3 by the application of both biochemical and genetic methods, we have been studying the factor in the yeast Saccharomyces cerevisiae. Yeast eIF3 was initially isolated and purified by a biochemical approach that used an eIF3-dependent mammalian assay for the synthesis of methionyl-puromycin (Met-PM) (17). The resulting yeast complex comprised eight subunits with apparent masses of 16,21, 29, 33, 39, 62, 90, and 135 kDa. The genes for three of the subunits had been identified previously, but it was not realized that they encode subunits of eIF3. The second-largest subunit, p90, is encoded by PRT1; the temperature-sensitive prt1-1 mutant causes destabilization of Met-tRNA i binding to 40S ribosomal subunits (8,15). GCD10, first characterized genetical...

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Characterization of Yeast Translation Initiation Factor 1A and Cloning of Its Essential Gene

Cited by 42 publications

References 36 publications

Mammalian Translation Initiation Factor eIF1 Functions with eIF1A and eIF3 in the Formation of a Stable 40 S Preinitiation Complex

Mammalian Translation Initiation Factor eIF1 Functions with eIF1A and eIF3 in the Formation of a Stable 40 S Preinitiation Complex

Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

The 39-Kilodalton Subunit of Eukaryotic Translation Initiation Factor 3 Is Essential for the Complex’s Integrity and for Cell Viability in Saccharomyces cerevisiae

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