ABSTRACT
Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5'UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2ß and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.
Subject(s)
Eukaryotic Initiation Factor-2B/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Peptide Initiation Factors/metabolism , Protein Biosynthesis , Ribosomes/metabolism , HEK293 Cells , Humans , RNA CapsABSTRACT
Death-associated protein 5 (DAP5/p97) is a homolog of the eukaryotic initiation factor 4G (eIF4G) that promotes the IRES-driven translation of multiple cellular mRNAs. Central to its function is the middle domain (MIF4G), which recruits the RNA helicase eIF4A. The middle domain of eIF4G consists of tandem HEAT repeats that coalesce to form a solenoid-type structure. Here, we report the crystal structure of the DAP5 MIF4G domain. Its overall fold is very similar to that of eIF4G; however, significant conformational variations impart distinct surface properties that could explain the observed differences in IRES binding between the two proteins. Interestingly, quantitative analysis of the DAP5-eIF4A interaction using isothermal titration calorimetry reveals a 10-fold lower affinity than with the eIF4G-eIF4A interaction that appears to affect their ability to stimulate eIF4A RNA unwinding activity in vitro. This difference in stability of the complex may have functional implications in selecting the mode of translation initiation.
Subject(s)
Eukaryotic Initiation Factor-4A/chemistry , Eukaryotic Initiation Factor-4G/chemistry , Models, Molecular , Protein Biosynthesis/genetics , Protein Conformation , Amino Acid Sequence , Binding Sites/genetics , Chromatography, Gel , Cloning, Molecular , Crystallization , Eukaryotic Initiation Factor-4A/genetics , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , Humans , Molecular Sequence Data , Mutagenesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Death-associated protein 5 (DAP5) is a member of the eIF4G family of scaffolding proteins that mediate cap-independent translation initiation by recruiting the translational machinery to internal ribosomal entry sites (IRESs) on mRNA. The MIF4G domain of DAP5 directly interacts with the eukaryotic initiation factors eIF4A and eIF3 and enhances the translation of several viral and cellular IRESs. Here, the crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 is presented.
