scholarly journals Polyribosome Binding by GCN1 Is Required for Full Activation of Eukaryotic Translation Initiation Factor 2α Kinase GCN2 during Amino Acid Starvation

2005 ◽  
Vol 280 (16) ◽  
pp. 16514-16521 ◽  
Author(s):  
Evelyn Sattlegger ◽  
Alan G. Hinnebusch
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Control Of Gene Expression ◽  
Eukaryotic Translation ◽  
Ribosome Binding ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation

The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2α. InSaccharomyces cerevisiae,activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1·GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1·GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1·GCN20 and a stronger decrease in eukaryotic translation initiation factor 2α phosphorylation than did mutating in one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells.

scholarly journals A Novel Function of the MA-3 Domains in Transformation and Translation Suppressor Pdcd4 Is Essential for Its Binding to Eukaryotic Translation Initiation Factor 4A

2004 ◽  
Vol 24 (9) ◽  
pp. 3894-3906 ◽  
Author(s):  
Hsin-Sheng Yang ◽  
Myung-Haing Cho ◽  
Halina Zakowicz ◽  
Glenn Hegamyer ◽  
Nahum Sonenberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Binding Activity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation Initiation Factor ◽  
Stem Loop ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Αn α-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4E249K, Pdcd4D253A, Pdcd4D414K, Pdcd4D418A, Pdcd4E249K,D414K, and Pdcd4D253A,D418A) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4D253A and Pdcd4D253A,D418A do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4D253A, Pdcd4D418A, or Pdcd4D235A,D418A. Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

scholarly journals Transcript Levels of the Eukaryotic Translation Initiation Factor 5A Gene Peak at Early G1 Phase of the Cell Cycle in the Dinoflagellate Crypthecodinium cohnii

2002 ◽  
Vol 68 (5) ◽  
pp. 2278-2284 ◽  
Author(s):  
K. L. Chan ◽  
D. New ◽  
S. Ghandhi ◽  
F. Wong ◽  
C. M. C. Lam ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Crypthecodinium Cohnii ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT A cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate Crypthecodinium cohnii (CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized C. cohnii cells, the transcript level peaked at early G1 and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (Saccharomyces cerevisiae) and certain mammalian cell types suggest a role for eIF-5A in the G1/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while falling within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor d-difluoromethylornithine and the specific hypusination inhibitor N-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinoflagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed.

scholarly journals Global Gene Expression Profiling Reveals Widespread yet Distinctive Translational Responses to Different Eukaryotic Translation Initiation Factor 2B-Targeting Stress Pathways

2005 ◽  
Vol 25 (21) ◽  
pp. 9340-9349 ◽  
Author(s):  
Julia B. Smirnova ◽  
Julian N. Selley ◽  
Fatima Sanchez-Cabo ◽  
Kathleen Carroll ◽  
A. Alan Eddy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Stress Conditions ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.

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