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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.


Peptidyl-tRNA hydrolase assay of the recombinant human L36AL protein using N-acetyl[3H]Phe-tRNAPhe as a substrate. Thepercent of residual N-acetyl[3H]Phe-tRNAPhe precipitable in trichloracetic acid was measured as a function of the concentration of therecombinant human RPL36AL. As a control, the activity of E. coli peptidyl-tRNA hydrolase (Pth) as a function of enzyme concentration isalso shown.
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Figure 10: Peptidyl-tRNA hydrolase assay of the recombinant human L36AL protein using N-acetyl[3H]Phe-tRNAPhe as a substrate. Thepercent of residual N-acetyl[3H]Phe-tRNAPhe precipitable in trichloracetic acid was measured as a function of the concentration of therecombinant human RPL36AL. As a control, the activity of E. coli peptidyl-tRNA hydrolase (Pth) as a function of enzyme concentration isalso shown.

Mentions: The presence of the unusually reactive Lys-53 in proximity to the universally conserved methylated GGQ motif common to translation termination factors (RFs) tempted us to speculate that the GGQ motif of RPL36AL is implicated in promoting peptidyl-tRNA hydrolysis as does the same motif in the RFs [19-21] and in proteins that display peptidyl-tRNA hydrolysis activity [22-24]. We addressed this question by measuring the peptidyl-tRNA hydrolysis activity of isolated recombinant RPL36AL, in comparison with that of the enzyme peptidyl-tRNA hydrolase (Pth) from E. coli, by using N-acetyl[3H]Phe-tRNAPhe as a substrate. As shown in Fig. (10), no Pth-like peptidyl-tRNA hydrolase activity was detectable under experimental conditions used for the Pth enzyme.


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)

Peptidyl-tRNA hydrolase assay of the recombinant human L36AL protein using N-acetyl[3H]Phe-tRNAPhe as a substrate. Thepercent of residual N-acetyl[3H]Phe-tRNAPhe precipitable in trichloracetic acid was measured as a function of the concentration of therecombinant human RPL36AL. As a control, the activity of E. coli peptidyl-tRNA hydrolase (Pth) as a function of enzyme concentration isalso shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Peptidyl-tRNA hydrolase assay of the recombinant human L36AL protein using N-acetyl[3H]Phe-tRNAPhe as a substrate. Thepercent of residual N-acetyl[3H]Phe-tRNAPhe precipitable in trichloracetic acid was measured as a function of the concentration of therecombinant human RPL36AL. As a control, the activity of E. coli peptidyl-tRNA hydrolase (Pth) as a function of enzyme concentration isalso shown.
Mentions: The presence of the unusually reactive Lys-53 in proximity to the universally conserved methylated GGQ motif common to translation termination factors (RFs) tempted us to speculate that the GGQ motif of RPL36AL is implicated in promoting peptidyl-tRNA hydrolysis as does the same motif in the RFs [19-21] and in proteins that display peptidyl-tRNA hydrolysis activity [22-24]. We addressed this question by measuring the peptidyl-tRNA hydrolysis activity of isolated recombinant RPL36AL, in comparison with that of the enzyme peptidyl-tRNA hydrolase (Pth) from E. coli, by using N-acetyl[3H]Phe-tRNAPhe as a substrate. As shown in Fig. (10), no Pth-like peptidyl-tRNA hydrolase activity was detectable under experimental conditions used for the Pth enzyme.

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.