title: Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome.
abstract: Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
authors.forename: Konstantin N
authors.initials: KN
authors.lastname: Bulygin
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
authors.forename: Yulia S
authors.initials: YS
authors.lastname: Bartuli
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Malygin
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
authors.forename: Dmitri M
authors.initials: DM
authors.lastname: Graifer
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
authors.forename: Ludmila Yu
authors.initials: LY
authors.lastname: Frolova
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
authors.forename: Galina G
authors.initials: GG
authors.lastname: Karpova
mesh_terms: D001665:Binding Sites; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D012346:RNA, Transfer, Amino Acyl; C087971:peptide-chain-release factor 3; C019037:tRNA, peptidyl-; D006160:Guanosine Triphosphate
keywords: chemical footprinting of rRNAs; human ribosome; rearrangements in rRNAs; release factors eRF1 and eRF3; translation termination