title: Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes.
abstract: 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 factor2alpha (eIF2alpha) 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.
authors.affiliation: Department of Microbiology and Immunology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Pisarev
authors.forename: Victoria G
authors.initials: VG
authors.lastname: Kolupaeva
authors.forename: Marat M
authors.initials: MM
authors.lastname: Yusupov
authors.forename: Christopher U T
authors.initials: CU
authors.lastname: Hellen
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Pestova
mesh_terms: D000818:Animals; D001483:Base Sequence; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D051379:Mice; D010442:Peptide Chain Initiation, Translational; D011487:Protein Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D013891:Thiouridine / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D039621:Eukaryotic Initiation Factor-3; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D013891:Thiouridine