title: Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome.
abstract: Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA(Asp) analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3'-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva, 8, 630090 Novosibirsk, Russia.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Bulygin
authors.forename: Alexey
authors.initials: A
authors.lastname: Malygin
authors.forename: Codjo
authors.initials: C
authors.lastname: Hountondji
authors.forename: Dmitri
authors.initials: D
authors.lastname: Graifer
authors.forename: Galina
authors.initials: G
authors.lastname: Karpova
mesh_terms: D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D003432:Cross-Linking Reagents; D020285:Cryoelectron Microscopy; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012339:RNA, Ribosomal, 28S; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013891:Thiouridine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D012339:RNA, Ribosomal, 28S; D012349:RNA, Transfer, Asp; D013891:Thiouridine