Abstract
Mammalian eIF3 is a 700-kDa multiprotein complex essential for initiation of protein synthesis in eukaryotic cells. It consists of 13 subunits (eIF3a to -m), among which eIF3b serves as a major scaffolding protein. Here we report the solution structure of the N-terminal RNA recognition motif of human eIF3b (eIF3b-RRM) determined by NMR spectroscopy. The structure reveals a noncanonical RRM with a negatively charged surface in the beta-sheet area contradictory with potential RNA binding activity. Instead, eIF3j, which is required for stable 40 S ribosome binding of the eIF3 complex, specifically binds to the rear alpha-helices of the eIF3b-RRM, opposite to its beta-sheet surface. Moreover, we identify that an N-terminal 69-amino acid peptide of eIF3j is sufficient for binding to eIF3b-RRM and that this interaction is essential for eIF3b-RRM recruitment to the 40 S ribosomal subunit. Our results provide the first structure of an important subdomain of a core eIF3 subunit and detailed insights into protein-protein interactions between two eIF3 subunits required for stable eIF3 recruitment to the 40 S subunit.
Highlights
Protein synthesis is crucial for the survival and propagation of life and represents one of the most complex and central events in the life cycle of eukaryotic cells
All of the experiments were performed at 25 °C. 7-l aliquots of domain, was overexpressed, isotopically eIF3b-RNA recognition motif (RRM) solution were injected into an eIF3j solution every labeled, and purified
The eIF3b-RRM lacks aromatic residues in the RNP2 sequence, and both RNPs are surrounded by acidic residues, rendering the RNA-binding interface in the -sheet area highly negatively charged (Fig. 1C), which is contradictory with RNA recognition and binding
Summary
Preparation of Recombinant Proteins—A DNA fragment encoding the eIF3b-RRM (residues 170 –274 of the human eIF3b subunit) was prepared by PCR from cDNA libraries (Clontech) and subcloned into pET28a vector (Novagen). A DNA fragment encoding the eIF3j subunit with a deletion of 6 of 7 nonconserved N-terminal alanine residues to eliminate the GC-rich coding sequence and enable preparation by PCR from cDNA libraries (Clontech), was subcloned into pET28a vector (Novagen) as His6-tagged fusion protein. To estimate the dependence of salt in the complex formation, the experiments were carried out with protein samples in buffer containing 0.4 M NaCl. Gel Mobility Shift Assays—Purified eIF3j (30 M final concentration) was incubated with increasing amount of purified eIF3b-RRM (7.5, 15, 22.5, 30, or 60 M final concentration) in a final volume of 20 l containing 10 mM Tris-HCl buffer (pH 7.4) and 50 mM NaCl.Complexes were formed at room temperature and analyzed on 8% native polyacrylamide gels.
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