scholarly journals Cloning of cDNA for the γ-subunit of mammalian translation initiation factor 2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2

1996 ◽  
Vol 318 (2) ◽  
pp. 631-636 ◽  
Author(s):  
Nigel T PRICE ◽  
Scot R. KIMBALL ◽  
Leonard S. JEFFERSON ◽  
Christopher G. PROUD
Keyword(s):  
Sequence Data ◽  
Pcr Primers ◽  
Genome Project ◽  
Translation Initiation Factor ◽  
Peptide Sequence ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Reading Frame ◽  
C Elegans ◽  
Γ Subunit

Peptide sequence data were obtained from rabbit protein synthesis initiation factor subunit eIF2Bγ. Searching the database of expressed sequence tags (dbEST) revealed nucleotide sequences potentially encoding human eIF2Bγ that contained peptides corresponding to those from the rabbit subunit. PCR primers were derived from these sequences and used to generate a probe. This was used to screen a rat skeletal muscle cDNA library, and a clone encoding rat eIF2Bγ was isolated. This cDNA gave a product in coupled transcription/translation that co-migrated with the γ-subunit of purified eIF2B under SDS/PAGE. The sequence of this rat eIF2Bγ cDNA is reported. The protein sequence shows homology with that of yeast eIF2Bγ (the GCD1 gene product). We have also identified an open reading frame from the Caenorhabditis elegans genome project that probably encodes the γ-subunit of C. elegans eIF2B. All these sequences show similarity to nucleotidyl- and acyltransferases, as previously reported for GCD1 [Koonin (1995) Protein Sci. 4, 1608–1617], and contain conserved motifs potentially involved in nucleotide binding. They also contain ‘I-patch’ motifs: isoleucine-rich hexamer repeats that have been associated with the binding of acyl groups in bacterial acyltransferases. The roles of these motifs are discussed in relation to the known properties of eIF2B.

scholarly journals Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1920-1932 ◽  
Author(s):  
J L Bushman ◽  
A I Asuru ◽  
R L Matts ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Open Reading Frames ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Molecular Weight Complex ◽  
Exchange Factor

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.

Conformational transitions in the γ subunit of the archaeal translation initiation factor 2

2014 ◽  
Vol 70 (3) ◽  
pp. 658-667 ◽  
Author(s):  
Oleg Nikonov ◽  
Elena Stolboushkina ◽  
Valentina Arkhipova ◽  
Olesya Kravchenko ◽  
Stanislav Nikonov ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Inorganic Phosphate ◽  
Bound States ◽  
Sulfolobus Solfataricus ◽  
Conformational Transitions ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Γ Subunit ◽  
Initiation Factor 2 ◽  
Translation Initiation Factor 2

In eukaryotes and archaea, the heterotrimeric translation initiation factor 2 (e/aIF2) is pivotal for the delivery of methionylated initiator tRNA (Met-tRNAi) to the ribosome. It acts as a molecular switch that cycles between inactive (GDP-bound) and active (GTP-bound) states. Recent studies show that eIF2 can also exist in a long-lived eIF2γ–GDP–Pi(inorganic phosphate) active state. Here, four high-resolution crystal structures of aIF2γ fromSulfolobus solfataricusare reported: aIF2γ–GDPCP (a nonhydrolyzable GTP analogue), aIF2γ–GDP–formate (in which a formate ion possibly mimics Pi), aIF2γ–GDP and nucleotide-free aIF2γ. The structures describe the different states of aIF2γ and demonstrate the conformational transitions that take place in the aIF2γ `life cycle'.

scholarly journals Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.

1993 ◽  
Vol 13 (8) ◽  
pp. 4618-4631 ◽  
Author(s):  
J L Bushman ◽  
M Foiani ◽  
A M Cigan ◽  
C J Paddon ◽  
A G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

scholarly journals Identification of Domains and Residues within the ɛ Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2Bɛ) Required for Guanine Nucleotide Exchange Reveals a Novel Activation Function Promoted by eIF2B Complex Formation

2000 ◽  
Vol 20 (11) ◽  
pp. 3965-3976 ◽  
Author(s):  
Edith Gomez ◽  
Graham D. Pavitt
Keyword(s):  
Catalytic Activity ◽  
Complex Formation ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
The Other ◽  
Initiation Factor ◽  
Missense Mutations ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bɛ). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bɛ in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiaegene encoding eIF2Bɛ (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bɛ (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bɛ without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bɛ disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bɛ alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bɛ alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bɛ and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bɛ is an activation domain that responds to eIF2B complex formation.

scholarly journals eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

2020 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

AbstractThe integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

scholarly journals eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

scholarly journals Oestrogen Sulfotransferase: Molecular Cloning and Sequencing of cDNA for the Bovine Placental Enzyme

1988 ◽  
Vol 41 (4) ◽  
pp. 507 ◽  
Author(s):  
Allan R Nash ◽  
Wendy K Glenn ◽  
Stephen S Moore ◽  
Judith Kerr ◽  
Adrienne R Thompson ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Sequence Data ◽  
Apparent Molecular Weight ◽  
Peptide Sequence ◽  
Binding Region ◽  
Cloning And Sequencing ◽  
Reading Frame ◽  
Physico Chemical ◽  
Malignant State ◽  
Steroid Binding

The female sex hormone, oestrogen, plays a central role in breast cell proliferation in both the normal and malignant state. It controls transcription from several genes, including that for the progesterone receptor, and in endometrial tissue, via this receptor, it controls the gene for the enzyme oestrogen sulfotransferase. This enzyme may control the level of the oestrogen receptor by sulfurylating free oestradiol. To study the mode of transcriptional control exercised by oestrogen, bovine oestrogen sulfotransferase cDNA has been cloned and the nucleotide sequence determined. The message, of which 1812 bases have been sequenced, contains an open reading frame of 885 bases which encode a protein of 295 amino acids and a maximum apparent molecular weight of 34 600. The deduced protein sequence is supported by existing peptide sequence data and appears to contain a steroid-binding region. Some physico-chemical characteristics of the enzyme appear to differ markedly from those previously reported.

scholarly journals Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3

1993 ◽  
Vol 13 (8) ◽  
pp. 4618-4631
Author(s):  
J L Bushman ◽  
M Foiani ◽  
A M Cigan ◽  
C J Paddon ◽  
A G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

scholarly journals Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J. Zachery Cogan ◽  
Rosalie E. Lawrence ◽  
Morgane Boone ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Double Stranded Rna ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2

AbstractViral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.

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