Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

Deoxyhypusine synthase catalyses the NAD-dependent transfer of the butylamine moiety from the polyamine, spermidine, to a specific lysine residue of a single cellular protein, eukaryotic translation-initiation factor 5A (eIF5A) precursor. The native enzyme exists as a tetramer of four identical subunits and contains four binding sites for NAD. The binding of spermidine and NAD was studied by a filtration assay. [3H]Spermidine binding to the enzyme was not detectable alone or in the presence of the eIF5A precursor, but was detected only in the presence of NAD or NADH, suggesting that a NAD/NADH-induced conformational change is required for the binding of spermidine. A strong NAD-dependent binding was also observed with a spermidine analogue, N1-guanyl-1,7-diamino[3H]heptane (GC7), but not with [14C]putrescine or [14C]spermine. Although [3H]NAD binding to the enzyme occurred in the absence of spermidine, its affinity for the enzyme was markedly enhanced by spermidine, GC7 and also by the eIF5A precursor. The maximum binding for NAD and spermidine was estimated to be ≈ 4 molecules each/enzyme tetramer. The dependence of spermidine binding on NAD and the modulation of binding of NAD by spermidine and the eIF5A precursor suggest intricate relationships between the binding of cofactor and the substrates, and provide new insights into the reaction mechanism.

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Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy

Protein Science ◽

10.1110/ps.18201 ◽

2008 ◽

Vol 10 (12) ◽

pp. 2426-2438 ◽

Cited By ~ 1

Author(s):

Wei Li ◽

David W. Hoffman

Keyword(s):

Nmr Spectroscopy ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Methanococcus Jannaschii ◽

Structure And Dynamics

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

Journal of Biological Chemistry ◽

10.1074/jbc.m414566200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 16514-16521 ◽

Cited By ~ 44

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.

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Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy

Protein Science ◽

10.1110/ps.ps.18201 ◽

2001 ◽

Vol 10 (12) ◽

pp. 2426-2438 ◽

Cited By ~ 15

Author(s):

W. Li

Keyword(s):

Nmr Spectroscopy ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Methanococcus Jannaschii ◽

Structure And Dynamics

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Back to translation: removal of aIF2 from the 5′-end of mRNAs by translation recovery factor in the crenarchaeon Sulfolobus solfataricus

Nucleic Acids Research ◽

10.1093/nar/gkt1169 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2505-2511 ◽

Cited By ~ 5

Author(s):

Birgit Märtens ◽

Salim Manoharadas ◽

David Hasenöhrl ◽

Lukas Zeichen ◽

Udo Bläsi

Keyword(s):

Stationary Phase ◽

Translation Initiation ◽

Sulfolobus Solfataricus ◽

Growth Conditions ◽

Translation Initiation Factor ◽

Recovery Factor ◽

Initiation Factor ◽

Ribosome Binding ◽

Γ Subunit

Abstract The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5′-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5′-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs. We have identified a protein termed Trf (translation recovery factor) that co-purified with trimeric aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of trimeric aIF2 from RNA, and that Trf directly interacts with the aIF2-γ subunit. The importance of Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant.

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Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.6940 ◽

1997 ◽

Vol 17 (12) ◽

pp. 6940-6947 ◽

Cited By ~ 191

Author(s):

H Imataka ◽

N Sonenberg

Keyword(s):

Amino Acid ◽

Binding Site ◽

Translation Initiation ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Amino Terminal ◽

Binding Domains ◽

Middle Domain ◽

Carboxy Terminal

Mammalian translation initiation factor 4F (eIF4F) consists of three subunits, eIF4A, eIF4E, and eIF4G. eIF4G interacts directly with both eIF4A and eIF4E. The binding site for eIF4E is contained in the amino-terminal third of eIF4G, while the binding site for eIF4A was mapped to the carboxy-terminal third of the molecule. Here we show that human eIF4G possesses two separate eIF4A binding domains in the middle third (amino acids [aa] 478 to 883) and carboxy-terminal third (aa 884 to 1404) of the molecule. The amino acid sequence of the middle portion of eIF4G is well conserved between yeasts and humans. We show that mutations of conserved amino acid stretches in the middle domain abolish or reduce eIF4A binding as well as eIF3 binding. In addition, a separate and nonoverlapping eIF4A binding domain exists in the carboxy-terminal third (aa 1045 to 1404) of eIF4G, which is not present in yeast. The C-terminal two-thirds region (aa 457 to 1404) of eIF4G, containing both eIF4A binding sites, is required for stimulating translation. Neither one of the eIF4A binding domains alone activates translation. In contrast to eIF4G, human p97, a translation inhibitor with homology to eIF4G, binds eIF4A only through the amino-terminal proximal region, which is homologous to the middle domain of eIF4G.

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Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding

Biochemistry ◽

10.1021/bi00163a005 ◽

1992 ◽

Vol 31 (48) ◽

pp. 11984-11990 ◽

Cited By ~ 26

Author(s):

Dominic De Bellis ◽

Dionysios Liveris ◽

Dixie Goss ◽

Steven Ringquist ◽

Ira Schwartz

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Translation Initiation ◽

Function Analysis ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Ribosome Binding

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Heteronuclear NMR studies of E. coli translation initiation factor IF3. evidence that the inter-domain region is disordered in solution 1 1 Edited by K. Nagai

Journal of Molecular Biology ◽

10.1006/jmbi.1996.0756 ◽

1997 ◽

Vol 266 (1) ◽

pp. 15-22 ◽

Cited By ~ 31

Author(s):

Magali Moreau ◽

Eve de Cock ◽

Pierre-Louis Fortier ◽

Carlos Garcia ◽

zChristine Albaret ◽

...

Keyword(s):

Translation Initiation ◽

Heteronuclear Nmr ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Nmr Studies ◽

E Coli

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Overlapping regions of Caf20 mediate its interactions with the mRNA-5′cap-binding protein eIF4E and with ribosomes

Scientific Reports ◽

10.1038/s41598-021-92931-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ebelechukwu C. Nwokoye ◽

Eiman AlNaseem ◽

Robert A. Crawford ◽

Lydia M. Castelli ◽

Martin D. Jennings ◽

...

Keyword(s):

Translation Initiation ◽

Selection Process ◽

Critical Role ◽

Interaction Region ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Ribosome Binding ◽

Binding Interface ◽

The Family ◽

Canonical Motif

AbstractBy interacting with the mRNA 5′ cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.

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