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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.


Model for the interactions between the ribosomal protein L36AL, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon.
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Figure 11: Model for the interactions between the ribosomal protein L36AL, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon.

Mentions: Now we are in the situation to propose a model for the interactions between the ribosomal L36AL protein, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon (Fig. 11), taking into account recently published structural and photocrosslinking data as well as the chemical crosslinking results reported here. We have recently proposed that the tRNAox species used in the present report might bind first to the classical P/P site before they flip spontaneously into the P/E site, where they are sampled by the crosslinking with Lys-53 of RPL36AL [7], in agreement with recent cryo-electron microscopy and single-molecule FRET data [33]. Our crosslinking data agree well with photocrosslinking data using a tRNAAsp analogue substituted with 4-thiouridine in position 76 (tRNAAspp-s4U76), which could be cross-linked to C4335 located very close to Lys-53 of RPL36AL in the E-site region of human 80S ribosome [18]. Crosslinking of the CCA-arm of a P-tRNA with both RPL36AL and eRF1 implies that the P-tRNA is present in the P/P state, while the L36AL protein due to the flexibility of its loop extension is capable of reaching the P-site, where it could interact with the CCA-arm and the acceptor stem of tRNA. Accordingly, we propose the model in Fig. (11), where the CCA end of the P-tRNA is sandwiched between the methylated GGQ motifs of RPL36AL and the A-site bound eRF1. In this model, the GGQ loop of eRF1 faces nucleotide A76 of P-tRNA, since at the end of the translation process, this nucleotide which bears the ester carbone of the peptidyl-tRNA is the natural target of the GGQ motif of eRF1 for the nucleophilic attack (via a water molecule) leading to the release of the polypeptide chain. Therefore, this model is compatible with the observation that the crosslinking yields of the ternary complexes “b” decrease as a consequence of the removal of A76 of the CCA-arm of tRNA. Decreasing of the crosslinking yield of the ternary complex “b” upon removal of A76 is in striking contrast to the crosslinking yields of the binary RPL36AL-tRNAox complexes “a”, which shows higher yields with tRNAox lacking the CCA-end. The latter results would suggest that the interaction with RPL36AL of the CCA region of tRNA is rather weak, and that other regions of the tRNA molecule are actually involved in the formation of a binary RPL36AL:tRNA complexes, as discussed above. One possible explanation is that, in the absence of eRF1, the CCA end of tRNA might be dispensable for the interaction between RPL36AL and the tRNA, while in its presence, the ribosomal protein makes specific contacts with the CCA end. This agrees well with the fact that the tRNA CCA-end must be in full-length for crosslinking both RPL36AL and eRF1.


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)

Model for the interactions between the ribosomal protein L36AL, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Model for the interactions between the ribosomal protein L36AL, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon.
Mentions: Now we are in the situation to propose a model for the interactions between the ribosomal L36AL protein, the CCA-arm of P-site bound tRNA and the translation termination factor eRF1 bound to an A-site stop codon (Fig. 11), taking into account recently published structural and photocrosslinking data as well as the chemical crosslinking results reported here. We have recently proposed that the tRNAox species used in the present report might bind first to the classical P/P site before they flip spontaneously into the P/E site, where they are sampled by the crosslinking with Lys-53 of RPL36AL [7], in agreement with recent cryo-electron microscopy and single-molecule FRET data [33]. Our crosslinking data agree well with photocrosslinking data using a tRNAAsp analogue substituted with 4-thiouridine in position 76 (tRNAAspp-s4U76), which could be cross-linked to C4335 located very close to Lys-53 of RPL36AL in the E-site region of human 80S ribosome [18]. Crosslinking of the CCA-arm of a P-tRNA with both RPL36AL and eRF1 implies that the P-tRNA is present in the P/P state, while the L36AL protein due to the flexibility of its loop extension is capable of reaching the P-site, where it could interact with the CCA-arm and the acceptor stem of tRNA. Accordingly, we propose the model in Fig. (11), where the CCA end of the P-tRNA is sandwiched between the methylated GGQ motifs of RPL36AL and the A-site bound eRF1. In this model, the GGQ loop of eRF1 faces nucleotide A76 of P-tRNA, since at the end of the translation process, this nucleotide which bears the ester carbone of the peptidyl-tRNA is the natural target of the GGQ motif of eRF1 for the nucleophilic attack (via a water molecule) leading to the release of the polypeptide chain. Therefore, this model is compatible with the observation that the crosslinking yields of the ternary complexes “b” decrease as a consequence of the removal of A76 of the CCA-arm of tRNA. Decreasing of the crosslinking yield of the ternary complex “b” upon removal of A76 is in striking contrast to the crosslinking yields of the binary RPL36AL-tRNAox complexes “a”, which shows higher yields with tRNAox lacking the CCA-end. The latter results would suggest that the interaction with RPL36AL of the CCA region of tRNA is rather weak, and that other regions of the tRNA molecule are actually involved in the formation of a binary RPL36AL:tRNA complexes, as discussed above. One possible explanation is that, in the absence of eRF1, the CCA end of tRNA might be dispensable for the interaction between RPL36AL and the tRNA, while in its presence, the ribosomal protein makes specific contacts with the CCA end. This agrees well with the fact that the tRNA CCA-end must be in full-length for crosslinking both RPL36AL and eRF1.

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.