Skip to Main content Skip to Navigation
Journal articles

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 Asite) 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 CCAarm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process
Document type :
Journal articles
Complete list of metadata

Cited literature [45 references]  Display  Hide  Download

https://hal.sorbonne-universite.fr/hal-01906405
Contributor : Gestionnaire Hal-Su Connect in order to contact the contributor
Submitted on : Friday, October 26, 2018 - 5:04:50 PM
Last modification on : Saturday, July 3, 2021 - 3:45:00 AM
Long-term archiving on: : Sunday, January 27, 2019 - 6:23:37 PM

File

6-TOBIOCJ-2014.pdf
Publication funded by an institution

Identifiers

Citation

Codjo Hountondji, Konstantin Bulygin, Jean-Bernard Crechet, Anne Woisard, Pierre Tuffery, et al.. 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. Open Biochemistry Journal, Bentham Open, 2014, 8, pp.52-67. ⟨10.2174/1874091X01408010052⟩. ⟨hal-01906405⟩

Share

Metrics

Record views

397

Files downloads

822