title: Arrangement of mRNA at the decoding site of human ribosomes. 18S rRNA nucleotides and ribosomal proteins cross-linked to oligouridylate derivatives with alkylating groups at either the 3' or the 5' termini.
abstract: Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphamide group at the 5'-termini or 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene residue attached to the 3'-termini, in the presence of cognate Phe-tRNA(Phe) has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5'-end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3'-termini, G1702 and G1763/G1764 were identified as the cross-linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross-linking sites of the mRNA analogs with the alkylating group at the 5'-termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12.
authors.affiliation: Laboratory of Ribosomal Structure and Functions, Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences.
authors.forename: A A
authors.initials: AA
authors.lastname: Malygin
authors.forename: D M
authors.initials: DM
authors.lastname: Graifer
authors.forename: K N
authors.initials: KN
authors.lastname: Bulygin
authors.forename: M A
authors.initials: MA
authors.lastname: Zenkova
authors.forename: V I
authors.initials: VI
authors.lastname: Yamkovoy
authors.forename: J
authors.initials: J
authors.lastname: Stahl
authors.forename: G G
authors.initials: GG
authors.lastname: Karpova
mesh_terms: D000345:Affinity Labels; D000478:Alkylation; D001596:Benzylamines; D003432:Cross-Linking Reagents; D005260:Female; D006801:Humans; D009693:Nucleic Acid Hybridization; D009843:Oligoribonucleotides / Q000378:metabolism*; D010920:Placenta / Q000648:ultrastructure; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014500:Uracil Nucleotides / Q000378:metabolism*
chemical_list: C091144:4-(N-2-chloroethyl-N-(methyl)amino)benzylmethylamine; D000345:Affinity Labels; D001596:Benzylamines; D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; D014500:Uracil Nucleotides; C027916:oligo(U)