Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases

1991 ◽  
Vol 11 (7) ◽  
pp. 3463-3471 ◽  
Author(s):  
S R Schmid ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Clear Correlation ◽  
Temperature Sensitive ◽  
Rna Helicases ◽  
Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

scholarly journals Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

1991 ◽  
Vol 11 (7) ◽  
pp. 3463-3471 ◽  
Author(s):  
S R Schmid ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Clear Correlation ◽  
Temperature Sensitive ◽  
Rna Helicases ◽  
Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

scholarly journals Eukaryotic Translation Initiation Factors 4G and 4A from Saccharomyces cerevisiae Interact Physically and Functionally

1999 ◽  
Vol 19 (8) ◽  
pp. 5557-5564 ◽  
Author(s):  
Carrie L. Neff ◽  
Alan B. Sachs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT The initiation of translation in eukaryotes requires several multisubunit complexes, including eukaryotic translation initiation factor 4F (eIF4F). In higher eukaryotes eIF4F is composed of the cap binding protein eIF4E, the adapter protein eIF4G, and the RNA-stimulated ATPase eIF4A. The association of eIF4A withSaccharomyces cerevisiae eIF4F has not yet been demonstrated, and therefore the degree to which eIF4A’s conserved function relies upon this association has remained unclear. Here we report an interaction between yeast eIF4G and eIF4A. Specifically, we found that the growth arrest phenotype associated with three temperature-sensitive alleles of yeast eIF4G2 was suppressed by excess eIF4A and that this suppression was allele specific. In addition, in vitro translation extracts derived from an eIF4G2 mutant strain could be heat inactivated, and this inactivation could be reversed upon the addition of recombinant eIF4A. Finally, in vitro binding between yeast eIF4G and eIF4A was demonstrated, as was diminished binding between mutant eIF4G2 proteins and eIF4A. In total, these data indicate that yeast eIF4G and eIF4A physically associate and that this association performs an essential function.

scholarly journals Eap1p, a Novel Eukaryotic Translation Initiation Factor 4E-Associated Protein in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (13) ◽  
pp. 4604-4613 ◽  
Author(s):  
Gregory P. Cosentino ◽  
Tobias Schmelzle ◽  
Ashkan Haghighat ◽  
Stephen B. Helliwell ◽  
Michael N. Hall ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Tor Signaling

ABSTRACT Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m7G(5′)ppp(5′)N, where N is any nucleotide] present at the 5′ termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of theEAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.

scholarly journals Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

1989 ◽  
Vol 9 (10) ◽  
pp. 4467-4472
Author(s):  
M Altmann ◽  
N Sonenberg ◽  
H Trachsel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Apparent Temperature ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Free System ◽  
Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

scholarly journals Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

1997 ◽  
Vol 17 (12) ◽  
pp. 6876-6886 ◽  
Author(s):  
S Z Tarun ◽  
A B Sachs
Keyword(s):  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Stimulation Of

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

scholarly journals Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

2007 ◽  
Vol 27 (6) ◽  
pp. 2384-2397 ◽  
Author(s):  
Jeanne M. Fringer ◽  
Michael G. Acker ◽  
Christie A. Fekete ◽  
Jon R. Lorsch ◽  
Thomas E. Dever
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

scholarly journals Implications of the Use of Eukaryotic Translation Initiation Factor 5A (eIF5A) for Prognosis and Treatment of Hepatocellular Carcinoma

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Felix H. Shek ◽  
Sarwat Fatima ◽  
Nikki P. Lee
Keyword(s):  
Hepatocellular Carcinoma ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) is a primary liver malignancy and accounts for most of the total liver cancer cases. Lack of treatment options and late diagnosis contribute to high mortality rate of HCC. In eukaryotes, translation of messenger RNA (mRNA) to protein is a key process in protein biosynthesis in which initiation of translation involves interaction of different eukaryotic translation initiation factors (eIFs), ribosome subunits and mRNAs. Eukaryotic translation initiation factor 5A (eIF5A) is one of the eIFs involved in translation initiation and eIF5A2, one of its isoforms, is upregulated in various cancers including HCC as a result of chromosomal instability, where it resides. In HCC, eIF5A2 expression is associated with adverse prognosis such as presence of tumor metastasis and venous infiltration. Based on eIF5A2 functional studies, suppressing eIF5A2 expression by short interfering RNA alleviates the tumorigenic properties of HCC cellsin vitrowhile ectopic expression of eIF5A2 enhances the aggressiveness of HCC cellsin vivoandin vitroby inducing epithelial-mesenchymal transition. In conclusion, eIF5A2 is a potential prognostic marker as well as a therapeutic target for HCC.

scholarly journals Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)

2013 ◽  
Vol 289 (3) ◽  
pp. 1704-1722 ◽  
Author(s):  
Fujun Zhou ◽  
Sarah E. Walker ◽  
Sarah F. Mitchell ◽  
Jon R. Lorsch ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Rna Recognition Motif ◽  
Conserved Motifs ◽  
Terminal Domain ◽  
Sequence Elements

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

scholarly journals Interaction of the RNP1 Motif in PRT1 with HCR1 Promotes 40S Binding of Eukaryotic Initiation Factor 3 in Yeast

2006 ◽  
Vol 26 (8) ◽  
pp. 2984-2998 ◽  
Author(s):  
Klaus H. Nielsen ◽  
Leos Valášek ◽  
Caroah Sykes ◽  
Antonina Jivotovskaya ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Eukaryotic Initiation Factor ◽  
Wild Type ◽  
Leaky Scanning ◽  
Rrm Domain ◽  
Subunit B

ABSTRACT We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts− phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts− phenotype is exacerbated by tif32-Δ6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Δ impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.

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