Several subunits of the multifunctional eukaryotic translation initiation factor 3 (eIF3) contain well-defined domains. Among them is the conserved bipartite PCI domain, typically serving as the principal scaffold for multisubunit 26S proteasome lid, CSN and eIF3 complexes, which constitutes most of the C-terminal region of the c/NIP1 subunit. Interestingly, the c/NIP1-PCI domain is exceptional in that its deletion, despite being lethal, does not affect eIF3 integrity. Here, we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 and eIF5 in vivo. The extreme C-terminus directly interacts with blades 1-3 of the small ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of the ASC1 coding region likewise affects eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this typical protein-protein binding domain in mediating protein-RNA interactions also. Importantly, as our clustered mutations severely reduce RNA binding, we conclude that the c/NIP1 C-terminal region forms an important intermolecular bridge between eIF3 and the 40S head region by contacting RACK1/ASC1 and most probably 18S rRNA.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• delece genu MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce MeSH
• interakční proteinové domény a motivy MeSH
• malé podjednotky ribozomu eukaryotické chemie   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající GTP chemie   genetika   metabolismus   MeSH
• RNA ribozomální 18S metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny biosyntéza   chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• transkripční faktory bZIP biosyntéza   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce fyziologie   MeSH
• kodon iniciační genetika   metabolismus   MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH