Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes

• Published in: The EMBO journal 2008-06, Vol.27 (11), p.1609-1621
• Main Authors: Pisarev, Andrey V, Kolupaeva, Victoria G, Yusupov, Marat M, Hellen, Christopher UT, Pestova, Tatyana V
• Format: Article
• Language: English
• Subjects: 4-thiouridine; Animals; Base Sequence; Binding sites; eIF3; EMBO31; Eukaryotes; Eukaryotic Initiation Factor-3; Eukaryotic Initiation Factor-3 - chemistry; Eukaryotic Initiation Factor-3 - metabolism; Eukaryotic Initiation Factor-3 - radiation effects; Genetics; Humans; Life Sciences; Mice; Molecular biology; mRNA; Peptide Chain Initiation, Translational; Protein Conformation; Proteins; Ribonucleic acid; Ribosomal Proteins; Ribosomal Proteins - chemistry; Ribosomal Proteins - metabolism; Ribosomal Proteins - radiation effects; ribosome; Ribosomes; Ribosomes - chemistry; Ribosomes - metabolism; Ribosomes - radiation effects; RNA; RNA, Messenger; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA, Messenger - radiation effects; Thiouridine; Thiouridine - chemistry; translation initiation; Ultraviolet Rays; Upstream
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2008.90

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4‐thiouridines. Crosslinking of mRNA positions + 11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and + 9– + 11 and + 8– + 9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA‐binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P‐site, the proximity of positions − 3/ − 4 to rpS5(S7p) and h23b, − 6/ − 7 to rpS14(S11p), and − 8– − 11 to the 3′‐terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine–Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions + 7/ + 8 to the central region of h28, + 4/ + 5 to rpS15(S19p), and − 6 and − 7/ − 10 to eukaryote‐specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2α (eIF2α) contacts position − 3, and now report that eIF3 interacts with positions − 8– − 17, forming an extension of the mRNA‐binding channel that likely contributes to unique aspects of eukaryotic initiation.