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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.


Crosslinking of recombinant human RPL36AL with periodate-oxidized full length tRNA (tRNAAsp76ox, lane 1), or tRNAoxspecies that were shortened by one (tRNAAsp75ox, lane 2), two (tRNAAsp74ox, lane 3), three (tRNAAsp73ox, lane 4) or four nucleotides(tRNAAsp72ox, lane 5) from the 3’-end. The incubation mixtures were applied onto a 10% polyacrylamide gel run by 8 M Ureaelectrophoresis and colored with Stains all. The “a” bands represent the tRNAox species crosslinked with recombinant L36AL, while thelower bands represent the tRNA analogues, as verified by routinely performed control experiments on the full length tRNA molecule(tRNAAsp76), or the truncated tRNA species.
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Figure 3: Crosslinking of recombinant human RPL36AL with periodate-oxidized full length tRNA (tRNAAsp76ox, lane 1), or tRNAoxspecies that were shortened by one (tRNAAsp75ox, lane 2), two (tRNAAsp74ox, lane 3), three (tRNAAsp73ox, lane 4) or four nucleotides(tRNAAsp72ox, lane 5) from the 3’-end. The incubation mixtures were applied onto a 10% polyacrylamide gel run by 8 M Ureaelectrophoresis and colored with Stains all. The “a” bands represent the tRNAox species crosslinked with recombinant L36AL, while thelower bands represent the tRNA analogues, as verified by routinely performed control experiments on the full length tRNA molecule(tRNAAsp76), or the truncated tRNA species.

Mentions: The full length tRNA molecule (tRNAAsp76), or the truncated tRNA species mentioned in the preceding section were treated with sodium periodate and the obtained 2’,3’-dialdehyde derivatives (tRNAox) were used for the crosslinking studies with isolated recombinant RPL36AL. Equimolar amounts of human recombinant RPL36AL were incubated in the presence of tRNAox (200 picomoles each), followed by an analysis of the crosslinks on urea-PAGE. Fig. (3) shows that a covalent complex is formed (band “a”), with a crosslinking yield of practically 100%, except in the cases of tRNA76ox and tRNA75ox (lanes 1 and 2, respectively). In fact, during the polyacrylamide gel electrophoresis in urea (Fig. 3), only tRNAox (lower band) and the tRNAox-L36AL covalent complex (upper “a” band) with their 75 (or so) negative charges of the phosphate groups would migrate toward the (+) pole at the bottom of the gel, while the free L36AL protein which is very basic (pI 10.59) would migrate toward the (-) pole at the top of the gel. As a consequence, the free L36AL would not even penetrate the urea-PAGE gel. Therefore, the non-crosslinked proteins in lanes 1 and 2 should not be visible. The possibility that the ribosomal protein could migrate as an unspecific, non-covalent complex together with the tRNA owing to the high binding affinity was ruled out by an SDS-PAGE analysis: the RPL36AL-tRNAox crosslinks showed RPL36AL-tRNAox covalent complexes corresponding to the band “a” in Fig. (3). Furthermore, the band “a” in Fig. (3) migrated with about 40,000 Da (revealed by comparison with standard marker proteins) indicating a 1:1 covalent complex between the His-tagged recombinant RPL36AL (MW of 15,000 Da) and the tRNAox species (MW of 25,000 Da, on average) (results not shown). Finally, the crosslinking yield with tRNA76ox is weaker than with the shortened tRNAox species (Fig. 3), in agreement with previously reported crosslinking data with the endogenous ribosomal protein L36AL in situ [7].


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)

Crosslinking of recombinant human RPL36AL with periodate-oxidized full length tRNA (tRNAAsp76ox, lane 1), or tRNAoxspecies that were shortened by one (tRNAAsp75ox, lane 2), two (tRNAAsp74ox, lane 3), three (tRNAAsp73ox, lane 4) or four nucleotides(tRNAAsp72ox, lane 5) from the 3’-end. The incubation mixtures were applied onto a 10% polyacrylamide gel run by 8 M Ureaelectrophoresis and colored with Stains all. The “a” bands represent the tRNAox species crosslinked with recombinant L36AL, while thelower bands represent the tRNA analogues, as verified by routinely performed control experiments on the full length tRNA molecule(tRNAAsp76), or the truncated tRNA species.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Crosslinking of recombinant human RPL36AL with periodate-oxidized full length tRNA (tRNAAsp76ox, lane 1), or tRNAoxspecies that were shortened by one (tRNAAsp75ox, lane 2), two (tRNAAsp74ox, lane 3), three (tRNAAsp73ox, lane 4) or four nucleotides(tRNAAsp72ox, lane 5) from the 3’-end. The incubation mixtures were applied onto a 10% polyacrylamide gel run by 8 M Ureaelectrophoresis and colored with Stains all. The “a” bands represent the tRNAox species crosslinked with recombinant L36AL, while thelower bands represent the tRNA analogues, as verified by routinely performed control experiments on the full length tRNA molecule(tRNAAsp76), or the truncated tRNA species.
Mentions: The full length tRNA molecule (tRNAAsp76), or the truncated tRNA species mentioned in the preceding section were treated with sodium periodate and the obtained 2’,3’-dialdehyde derivatives (tRNAox) were used for the crosslinking studies with isolated recombinant RPL36AL. Equimolar amounts of human recombinant RPL36AL were incubated in the presence of tRNAox (200 picomoles each), followed by an analysis of the crosslinks on urea-PAGE. Fig. (3) shows that a covalent complex is formed (band “a”), with a crosslinking yield of practically 100%, except in the cases of tRNA76ox and tRNA75ox (lanes 1 and 2, respectively). In fact, during the polyacrylamide gel electrophoresis in urea (Fig. 3), only tRNAox (lower band) and the tRNAox-L36AL covalent complex (upper “a” band) with their 75 (or so) negative charges of the phosphate groups would migrate toward the (+) pole at the bottom of the gel, while the free L36AL protein which is very basic (pI 10.59) would migrate toward the (-) pole at the top of the gel. As a consequence, the free L36AL would not even penetrate the urea-PAGE gel. Therefore, the non-crosslinked proteins in lanes 1 and 2 should not be visible. The possibility that the ribosomal protein could migrate as an unspecific, non-covalent complex together with the tRNA owing to the high binding affinity was ruled out by an SDS-PAGE analysis: the RPL36AL-tRNAox crosslinks showed RPL36AL-tRNAox covalent complexes corresponding to the band “a” in Fig. (3). Furthermore, the band “a” in Fig. (3) migrated with about 40,000 Da (revealed by comparison with standard marker proteins) indicating a 1:1 covalent complex between the His-tagged recombinant RPL36AL (MW of 15,000 Da) and the tRNAox species (MW of 25,000 Da, on average) (results not shown). Finally, the crosslinking yield with tRNA76ox is weaker than with the shortened tRNAox species (Fig. 3), in agreement with previously reported crosslinking data with the endogenous ribosomal protein L36AL in situ [7].

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.