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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.

Hountondji C, Bulygin K, Créchet JB, Woisard A, Tuffery P, Nakayama J, Frolova L, Nierhaus KH, Karpova G, Baouz S - Open Biochem J (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Kinetic measurement of RPL36AL:tRNA interaction. (A), His-tagged RPL36AL was immobilized on the surface of NTA sensor chip at low density resonance units (RU). Various concentrations of tRNAAsp76 (0, 1, 2, 4, 5 and 6 nM) were run over the chip surface. (B), the same experiment with tRNAAsp71.
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Figure 2: Kinetic measurement of RPL36AL:tRNA interaction. (A), His-tagged RPL36AL was immobilized on the surface of NTA sensor chip at low density resonance units (RU). Various concentrations of tRNAAsp76 (0, 1, 2, 4, 5 and 6 nM) were run over the chip surface. (B), the same experiment with tRNAAsp71.

Mentions: The possible interactions between a P-tRNA and the protein L36AL, and the similarity to tRNA•synthetase interactions prompted us to test whether isolated recombinant L36AL can bind tRNA. We used the Biacore assay, which allows an easy assessment of the strength of a possible interaction. To this end, we used various tRNA fragments for the binding assays. In addition to the full length tRNA (tRNAAsp76), we tested tRNA species that were shortened by one (tRNAAsp75), two (tRNAAsp74), three (tRNAAsp73), four (tRNAAsp72) or five (tRNAAsp71) nucleotides from the 3’-end. To determine the binding affinities of RPL36AL to tRNA variants, we carried out surface plasmon resonance (SPR) analyses with a Biacore biosensor, which provides real-time protein:RNA interaction data by measuring the change in refractive index at the surface of the sensor chip due to the interactions (Figs. 2A and 2B). Experimental data from individual kinetic binding experiments were analyzed and fitted using BIAevaluation software with 1:1 binding model, in accordance to the 1:1 stoichiometries of binding of tRNA to the aminoacyl-tRNA synthetases polypeptide chains [9,16].


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.

Hountondji C, Bulygin K, Créchet JB, Woisard A, Tuffery P, Nakayama J, Frolova L, Nierhaus KH, Karpova G, Baouz S - Open Biochem J (2014)

Kinetic measurement of RPL36AL:tRNA interaction. (A), His-tagged RPL36AL was immobilized on the surface of NTA sensor chip at low density resonance units (RU). Various concentrations of tRNAAsp76 (0, 1, 2, 4, 5 and 6 nM) were run over the chip surface. (B), the same experiment with tRNAAsp71.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150381&req=5

Figure 2: Kinetic measurement of RPL36AL:tRNA interaction. (A), His-tagged RPL36AL was immobilized on the surface of NTA sensor chip at low density resonance units (RU). Various concentrations of tRNAAsp76 (0, 1, 2, 4, 5 and 6 nM) were run over the chip surface. (B), the same experiment with tRNAAsp71.
Mentions: The possible interactions between a P-tRNA and the protein L36AL, and the similarity to tRNA•synthetase interactions prompted us to test whether isolated recombinant L36AL can bind tRNA. We used the Biacore assay, which allows an easy assessment of the strength of a possible interaction. To this end, we used various tRNA fragments for the binding assays. In addition to the full length tRNA (tRNAAsp76), we tested tRNA species that were shortened by one (tRNAAsp75), two (tRNAAsp74), three (tRNAAsp73), four (tRNAAsp72) or five (tRNAAsp71) nucleotides from the 3’-end. To determine the binding affinities of RPL36AL to tRNA variants, we carried out surface plasmon resonance (SPR) analyses with a Biacore biosensor, which provides real-time protein:RNA interaction data by measuring the change in refractive index at the surface of the sensor chip due to the interactions (Figs. 2A and 2B). Experimental data from individual kinetic binding experiments were analyzed and fitted using BIAevaluation software with 1:1 binding model, in accordance to the 1:1 stoichiometries of binding of tRNA to the aminoacyl-tRNA synthetases polypeptide chains [9,16].

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.