Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

Nucleic Acids Res. 2009 Aug;37(15):5167-82. doi: 10.1093/nar/gkp519. Epub 2009 Jun 26.

Abstract

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA(i)(Met) attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA(i)(Met), but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA(i)(Met) reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA(i)(Met), consistent with its suggested role in promoting the 'closed' conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the 'closed' complex and is likely ejected from the P-site upon start codon recognition.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Eukaryotic Initiation Factor-1 / chemistry*
  • Eukaryotic Initiation Factor-1 / genetics
  • Eukaryotic Initiation Factor-1 / metabolism
  • Hydroxyl Radical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Structure, Tertiary
  • RNA, Ribosomal, 18S / chemistry
  • Rabbits
  • Ribosome Subunits, Small, Eukaryotic / chemistry*
  • Ribosome Subunits, Small, Eukaryotic / metabolism

Substances

  • Eukaryotic Initiation Factor-1
  • RNA, Ribosomal, 18S
  • eukaryotic peptide initiation factor-1A
  • Hydroxyl Radical