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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.


Structure of the ternary RPL36AL-tRNAox-eRF1 covalent complex.
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Figure 6: Structure of the ternary RPL36AL-tRNAox-eRF1 covalent complex.

Mentions: We tried formaldehyde and glutaraldehyde as crosslinking reagents, but failed to detect crosslinks between the [32P]-labeled P-tRNAox and eRF1 or any ribosomal component. Since formaldehyde is dedicated to the crosslinking of macromolecules that are in close contact, failure of this reagent would suggest that the P-tRNA is not in a close contact with either the L36AL protein or the eRF1 at the A-site. An alternative possibility is that the absence of a crosslink could reflect the absence of suitable targets for crosslinking in the contact regions of these macromolecules. Surprisingly, bands whose electrophoretic mobility corresponded to distinct covalent complexes made of L36AL, tRNAox and eRF1 were observed on the SDS-PAGE gel in the absence of reagents (Fig. 5A, lane 1). These findings suggested that the two aldehyde groups of tRNAox react with both L36AL and eRF1 (Fig. 6). Comparison of the migration of the crosslinked complexes “a” and “b” to that of protein markers on the same gels indicated apparent molecular weights of 36,000 + 1,000 Da (“a”) and 87,000 + 2,000 Da (“b”). The molecular weight of the band “a” agreed surprisingly well with that predicted for a binary complex containing one molecule of endogenous RPL36AL (12,000 Da) and one of tRNAox (25,000 Da; calculated MW of the binary complex 37,000 Da). Likewise, the MW of complex “b” agreed well with a ternary complex that contained in addition to the binary one one molecule of eRF1 (48,000 Da) leading to a calculated MW of 85,000 Da (ternary complex RPL36AL-tRNAox-eRF1 “b”, see Figs. 5A, 5C and 6). We note that, when the crosslinking of human 80S ribosomes was performed with an elongator tRNA-Met-ox positioned at the P-site in the presence of an appropriate mRNA and of eRF1 bound to an A-site stop codon, the ternary complex “b” was formed with comparable crosslinking yields (results not shown) suggesting that both tRNA dialdehyde derivatives, when bound to the P-site, behave similarly in the crosslinking reaction.


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)

Structure of the ternary RPL36AL-tRNAox-eRF1 covalent complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Structure of the ternary RPL36AL-tRNAox-eRF1 covalent complex.
Mentions: We tried formaldehyde and glutaraldehyde as crosslinking reagents, but failed to detect crosslinks between the [32P]-labeled P-tRNAox and eRF1 or any ribosomal component. Since formaldehyde is dedicated to the crosslinking of macromolecules that are in close contact, failure of this reagent would suggest that the P-tRNA is not in a close contact with either the L36AL protein or the eRF1 at the A-site. An alternative possibility is that the absence of a crosslink could reflect the absence of suitable targets for crosslinking in the contact regions of these macromolecules. Surprisingly, bands whose electrophoretic mobility corresponded to distinct covalent complexes made of L36AL, tRNAox and eRF1 were observed on the SDS-PAGE gel in the absence of reagents (Fig. 5A, lane 1). These findings suggested that the two aldehyde groups of tRNAox react with both L36AL and eRF1 (Fig. 6). Comparison of the migration of the crosslinked complexes “a” and “b” to that of protein markers on the same gels indicated apparent molecular weights of 36,000 + 1,000 Da (“a”) and 87,000 + 2,000 Da (“b”). The molecular weight of the band “a” agreed surprisingly well with that predicted for a binary complex containing one molecule of endogenous RPL36AL (12,000 Da) and one of tRNAox (25,000 Da; calculated MW of the binary complex 37,000 Da). Likewise, the MW of complex “b” agreed well with a ternary complex that contained in addition to the binary one one molecule of eRF1 (48,000 Da) leading to a calculated MW of 85,000 Da (ternary complex RPL36AL-tRNAox-eRF1 “b”, see Figs. 5A, 5C and 6). We note that, when the crosslinking of human 80S ribosomes was performed with an elongator tRNA-Met-ox positioned at the P-site in the presence of an appropriate mRNA and of eRF1 bound to an A-site stop codon, the ternary complex “b” was formed with comparable crosslinking yields (results not shown) suggesting that both tRNA dialdehyde derivatives, when bound to the P-site, behave similarly in the crosslinking reaction.

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.