Folding transitions during assembly of the eukaryotic mRNA cap-binding complex.

The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.

[1]  U. Maitra,et al.  Regulation of GTP hydrolysis prior to ribosomal AUG selection during eukaryotic translation initiation , 2005, The EMBO journal.

[2]  Jon R Lorsch,et al.  The molecular mechanics of eukaryotic translation. , 2003, Annual review of biochemistry.

[3]  A. Sachs,et al.  Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. McCarthy,et al.  Cooperative modulation by eIF4G of eIF4E‐binding to the mRNA 5′ cap in yeast involves a site partially shared by p20 , 1998, The EMBO journal.

[5]  C. Berset,et al.  A novel inhibitor of cap‐dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E , 1997, The EMBO journal.

[6]  Anne-Claude Gingras,et al.  Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. , 2002, Journal of molecular biology.

[7]  H. Trachsel,et al.  Altered mRNA cap recognition activity of initiation factor 4E in the yeast cell cycle division mutant cdc33. , 1989, Nucleic acids research.

[8]  A. Gingras,et al.  Cocrystal Structure of the Messenger RNA 5′ Cap-Binding Protein (eIF4E) Bound to 7-methyl-GDP , 1997, Cell.

[9]  J. McCarthy,et al.  Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem‐loops in the mRNA leader , 1993, Molecular microbiology.

[10]  A. Gingras,et al.  Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. , 1999, Molecular cell.

[11]  G. Wagner,et al.  Ribosome Loading onto the mRNA Cap Is Driven by Conformational Coupling between eIF4G and eIF4E , 2003, Cell.

[12]  G. Wagner,et al.  The mRNA cap-binding protein eIF4E in post-transcriptional gene expression , 2004, Nature Structural &Molecular Biology.

[13]  A. Gingras,et al.  Structure of translation factor elF4E bound to m7GDP and interaction with 4E-binding protein , 1997, Nature Structural Biology.

[14]  N. Sonenberg,et al.  Translational control of gene expression , 2000 .

[15]  N. Sonenberg,et al.  Circular dichroism and fluorescence studies on protein synthesis initiation factor eIF-4E and two mutant forms from the yeast Saccharomyces cerevisiae. , 1988, The Journal of biological chemistry.

[16]  A. Kentsis,et al.  PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA , 2001, The EMBO journal.

[17]  K. Browning,et al.  pH-dependent and ligand induced conformational changes of eucaryotic protein synthesis initiation factor eIF-(iso)4F: a circular dichroism study. , 1996, Biochimica et biophysica acta.

[18]  H. Dyson,et al.  Intrinsically unstructured proteins and their functions , 2005, Nature Reviews Molecular Cell Biology.

[19]  J. McCarthy,et al.  Posttranscriptional Control of Gene Expression in Yeast , 1998, Microbiology and Molecular Biology Reviews.

[20]  J. McCarthy,et al.  Stabilization of Eukaryotic Initiation Factor 4E Binding to the mRNA 5′-Cap by Domains of eIF4G* , 2000, The Journal of Biological Chemistry.

[21]  A. Hinnebusch,et al.  A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator tRNA(Met) is an important translation initiation intermediate in vivo. , 2000, Genes & development.

[22]  I. Mikaélian,et al.  A general and fast method to generate multiple site directed mutations. , 1992, Nucleic acids research.

[23]  G. Wagner,et al.  The Cap-binding Protein eIF4E Promotes Folding of a Functional Domain of Yeast Translation Initiation Factor eIF4G1* , 1999, The Journal of Biological Chemistry.

[24]  K. Tomoo,et al.  Structural features of human initiation factor 4E, studied by X-ray crystal analyses and molecular dynamics simulations. , 2003, Journal of molecular biology.

[25]  J. Hershey,et al.  2 The Pathway and Mechanism of Initiation of Protein Synthesis , 2000 .