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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.


Analysis in 10% SDS-PAGE of crosslinked ribosomalcomplexes 1-5 obtained in the presence of [5’-32P]tRNAAsp76ox(lanes 1-5, respectively). Autoradiogram of the gel. The positionsof tRNA analogues and crosslinking products are marked.
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Figure 7: Analysis in 10% SDS-PAGE of crosslinked ribosomalcomplexes 1-5 obtained in the presence of [5’-32P]tRNAAsp76ox(lanes 1-5, respectively). Autoradiogram of the gel. The positionsof tRNA analogues and crosslinking products are marked.

Mentions: It is interesting to note that a recently published cryo-electron microscopy study of the mammalian eukaryotic eRF1-eRF3-associated termination complex revealed that eRF1 must undergo substantial conformational changes to accomodate the GGQ motif into the PTC [17]. To test this possibility in the context of the crosslinking of eRF1 with tRNAox positioned at the P-site, we constructed 80S ribosomal complexes containing, in addition to a tRNAAsp76ox at the P-site and the eRF1 factor at the A-site, eRF3 in association with its substrate GTP or with GDPNP (the non-hydrolyzable GTP analog guanosine 5’-[β,γ-imido]-triphosphate; see Fig. 7). Analysis in 10% SDS-PAGE of crosslinked “a” and “b” bands corresponding to the binary complex RPL36AL-tRNAox and the ternary complex RPL36AL-tRNAox-eRF1, respectively, revealed that the “b” band is quenched in the presence of eRF3 independently of the nature of the guanine nucleotide (GTP or GDPNP) used (compare lanes 4 and 5 with lane 3 in Fig. 7). These results are consistent with a conformational change of eRF1 upon eRF3 binding on the ribosome and with the observation that eRF3 does not leave the ribosome after GTP hydrolysis [17]. Furthermore, quenching of the “b” band in the presence of eRF3 reflects the formation of eRF1-eRF3-GTP complex, in which eRF1 adopts a different conformation, as compared with the situation in the absence of eRF3. All in all, regarding the crosslinking of tRNAox with eRF1 on one hand, and with L36AL or L37 on the other hand, one can imagine that the P/E tRNAox forms a crosslink with L36AL or L37, then rearranges before crosslinking with eRF1 which would be also subjected to some conformational change, as discussed above. Finally, for unknown reasons, the intensities of the “a” bands (RPL36AL-tRNAox complex) were also affected by the binding of eRF3 to eRF1 (compare lanes 4 and 5 with lane 3 in Fig. 7). Another observation from Fig. (7) is that the yields of crosslinking of “b” band containing the ternary complex RPL36AL-tRNAox-eRF1 is similar in the absence or presence of an E-site tRNA (compare lanes 2 and 3 in Fig. 7). As native tRNAAsp was recently shown to bind easily to the E-site of the human large ribosomal subunit [7], these results might be interpreted as follows: In the presence of an E-tRNA the neighboured tRNAox cannot adopt the hybrid state P/E but rather the classical P/P site. A crosslink of L36AL with tRNAox in this situation shows that this ribosomal protein lies closer to the P site than previously reported [7]. Finally, the human 80S ribosome in a complex with P-site bound tRNAox and with A-site bound eRF1 used here mimics the post-termination state of the ribosome. Thus the results shed light onto the mutual positioning of the P-tRNA, L36AL and eRF1 at the end of the translation process.


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)

Analysis in 10% SDS-PAGE of crosslinked ribosomalcomplexes 1-5 obtained in the presence of [5’-32P]tRNAAsp76ox(lanes 1-5, respectively). Autoradiogram of the gel. The positionsof tRNA analogues and crosslinking products are marked.
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Related In: Results  -  Collection

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

Figure 7: Analysis in 10% SDS-PAGE of crosslinked ribosomalcomplexes 1-5 obtained in the presence of [5’-32P]tRNAAsp76ox(lanes 1-5, respectively). Autoradiogram of the gel. The positionsof tRNA analogues and crosslinking products are marked.
Mentions: It is interesting to note that a recently published cryo-electron microscopy study of the mammalian eukaryotic eRF1-eRF3-associated termination complex revealed that eRF1 must undergo substantial conformational changes to accomodate the GGQ motif into the PTC [17]. To test this possibility in the context of the crosslinking of eRF1 with tRNAox positioned at the P-site, we constructed 80S ribosomal complexes containing, in addition to a tRNAAsp76ox at the P-site and the eRF1 factor at the A-site, eRF3 in association with its substrate GTP or with GDPNP (the non-hydrolyzable GTP analog guanosine 5’-[β,γ-imido]-triphosphate; see Fig. 7). Analysis in 10% SDS-PAGE of crosslinked “a” and “b” bands corresponding to the binary complex RPL36AL-tRNAox and the ternary complex RPL36AL-tRNAox-eRF1, respectively, revealed that the “b” band is quenched in the presence of eRF3 independently of the nature of the guanine nucleotide (GTP or GDPNP) used (compare lanes 4 and 5 with lane 3 in Fig. 7). These results are consistent with a conformational change of eRF1 upon eRF3 binding on the ribosome and with the observation that eRF3 does not leave the ribosome after GTP hydrolysis [17]. Furthermore, quenching of the “b” band in the presence of eRF3 reflects the formation of eRF1-eRF3-GTP complex, in which eRF1 adopts a different conformation, as compared with the situation in the absence of eRF3. All in all, regarding the crosslinking of tRNAox with eRF1 on one hand, and with L36AL or L37 on the other hand, one can imagine that the P/E tRNAox forms a crosslink with L36AL or L37, then rearranges before crosslinking with eRF1 which would be also subjected to some conformational change, as discussed above. Finally, for unknown reasons, the intensities of the “a” bands (RPL36AL-tRNAox complex) were also affected by the binding of eRF3 to eRF1 (compare lanes 4 and 5 with lane 3 in Fig. 7). Another observation from Fig. (7) is that the yields of crosslinking of “b” band containing the ternary complex RPL36AL-tRNAox-eRF1 is similar in the absence or presence of an E-site tRNA (compare lanes 2 and 3 in Fig. 7). As native tRNAAsp was recently shown to bind easily to the E-site of the human large ribosomal subunit [7], these results might be interpreted as follows: In the presence of an E-tRNA the neighboured tRNAox cannot adopt the hybrid state P/E but rather the classical P/P site. A crosslink of L36AL with tRNAox in this situation shows that this ribosomal protein lies closer to the P site than previously reported [7]. Finally, the human 80S ribosome in a complex with P-site bound tRNAox and with A-site bound eRF1 used here mimics the post-termination state of the ribosome. Thus the results shed light onto the mutual positioning of the P-tRNA, L36AL and eRF1 at the end of the translation process.

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.