Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome |
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Authors: | Konstantin Bulygin Alexey Malygin Codjo Hountondji Dmitri Graifer Galina Karpova |
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Institution: | 1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva, 8, 630090 Novosibirsk, Russia;2. UPMC Univ Paris 06, Equipe de Photobiologie Moléculaire, Tour 32 (4ème Etage, couloir 32-33), Case Courrier 60, 4 place Jussieu, F-75252 Paris Cedex 05, France |
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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 tRNAAsp 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. |
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Keywords: | 80S ribosomes tRNA binding sites CCA-end localization 4-Thiouridine photocross-linking 28S rRNA Hydroxyl radical footprinting |
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