title: The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome.
abstract: We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.
authors.affiliation: Sorbonne Universités UPMC Univ Paris 06, Unité de Recherche UPMC UR6 "Enzymologie de l'ARN", 2, Place Jussieu, F-75252 Paris Cedex 05, France.
authors.forename: Codjo
authors.initials: C
authors.lastname: Hountondji
authors.affiliation: Sorbonne Universités UPMC Univ Paris 06, Unité de Recherche UPMC UR6 "Enzymologie de l'ARN", 2, Place Jussieu, F-75252 Paris Cedex 05, France ; Institute of Chemical Biology and Fundamental Medecine, Siberian Branch of the Russian Academy of Sciences, pr Lavrentieva, 8, 630090 Novosibirsk, Russia.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Bulygin
authors.affiliation: Ecole Polytechnique, Route de Saclay, F-91120 Palaiseau, France.
authors.forename: Jean-Bernard
authors.initials: JB
authors.lastname: Créchet
authors.affiliation: Sorbonne Universités UPMC Univ Paris 06, Unité de Recherche UPMC UR6 "Enzymologie de l'ARN", 2, Place Jussieu, F-75252 Paris Cedex 05, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Woisard
authors.affiliation: Université Denis Diderot-Paris 7, INSERM-UMR-S973 and RPBS, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Tuffery
authors.affiliation: Graduate School of Natural Sciences, Nagoya City University, 1 Yamanohata, Mizuho, Nagoya, Aichi 467-8501, Japan.
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Nakayama
authors.affiliation: Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences, 119991 Moscow, Russia.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Frolova
authors.affiliation: Charité, Institut für Medizinische Physik und Biophysic, Charitéplatz 1. D-10117 Berlin, Germany.
authors.forename: Knud H
authors.initials: KH
authors.lastname: Nierhaus
authors.affiliation: Institute of Chemical Biology and Fundamental Medecine, Siberian Branch of the Russian Academy of Sciences, pr Lavrentieva, 8, 630090 Novosibirsk, Russia.
authors.forename: Galina
authors.initials: G
authors.lastname: Karpova
authors.affiliation: Sorbonne Universités UPMC Univ Paris 06, Unité de Recherche UPMC UR6 "Enzymologie de l'ARN", 2, Place Jussieu, F-75252 Paris Cedex 05, France.
authors.forename: Soria
authors.initials: S
authors.lastname: Baouz
keywords: A-site stop codon; CCA-end; P-tRNA; RPL36AL/tRNAox/eRF1 ternary complex on the human 80S ribosome.; abnormally low pK for Lys-53 of human RPL36AL; conformational change of eRF1 upon binding to the ribosome; crosslinking; eRF1; effect of the eRF1/eRF3 complex on the crosslinking of eRF1 in the human 80S ribosome; human s80S ribosomes; recombinant human RPL36AL