title: Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex.
abstract: Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at (-)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of (-)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C(1696) and less well to AA(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818-1819). U at (-)3 cross-linked to rpS5 and eIF2alpha, whereas G cross-linked only to eIF2alpha. Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha-subunit-deficient eIF2 indicate that eIF2alpha's interaction with the (-)3 purine is responsible for recognition of the (-)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818-1819) interaction might be responsible for recognizing the (+)4 position.
authors.affiliation: Department of Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, New York 11203, USA.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Pisarev
authors.forename: Victoria G
authors.initials: VG
authors.lastname: Kolupaeva
authors.forename: Vera P
authors.initials: VP
authors.lastname: Pisareva
authors.forename: William C
authors.initials: WC
authors.lastname: Merrick
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; D002417:Cattle; D018387:Codon, Initiator / Q000378:metabolism*; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013872:Thionucleosides / Q000737:chemistry / Q000378:metabolism*; D013891:Thiouridine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D039642:Eukaryotic Initiation Factors; D012333:RNA, Messenger; D013872:Thionucleosides; D006151:Guanosine; D013891:Thiouridine; C005783:6-thioguanosine