Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing.
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.
Yu
Yingpu
Y
Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA.
Marintchev
Assen
A
Kolupaeva
Victoria G
VG
Unbehaun
Anett
A
Veryasova
Tatyana
T
Lai
Shao-Chiang
SC
Hong
Peng
P
Wagner
Gerhard
G
Hellen
Christopher U T
CU
Pestova
Tatyana V
TV
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012337:RNA, Ribosomal, 18S / Q000737:chemistry; D011817:Rabbits; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry* / Q000378:metabolism
D015977:Eukaryotic Initiation Factor-1; D012337:RNA, Ribosomal, 18S; C026022:eukaryotic peptide initiation factor-1A; D017665:Hydroxyl Radical