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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 of the L36AL protein and of its interaction with tRNA. (A), ribbon representation of the 3-D structure of human RPL36AL(fragment 1-94) modeled by homology with the crystallographic structure of the archaeal counterpart RPL44E of the 60S ribosomal subunitfrom Haloarcula marismortui. The post-translational modifications (including the methylated Q51) [7], and the consensus pattern61Kx(TorV)KKxxL(KorR)xxC72 (numbering of human RPL36AL) of the L44e family of r-proteins are colored pink and cyan, respectively.The 49GG50 dipeptide of the GGQ motif is highlighted in green. A zinc ion represented by a cadmium colored yellow is also shown.Fragment 86-94 corresponding to the nucleotide binding motif 2 (NBD2) common to all eukaryotic RPL7 [29] and RPL36A/RPL44 is shownin wheat. (B), overlaid structures of tRNAPhe (PDB ID 1JGQ) colored grey and of human RPL36AL (blue). The GGQ motif is shown in red,the 3’ terminal CCA trinucleotide of tRNA in pink, and the side chain of Lys-53 in green. (C), NBD2 [29] located in the C-terminal region ofthe eukaryotic RPL7 family (ortholog of the bacterial L30 protein) is conserved in the C-terminal region of the eukaryotic RPL36A/RPL44family. The organisms are: Arabidopsis thaliana (arb. thal.), Drosophila melanogaster (droso.), Saccharomyces cerevisiae (s. cer.), Solanumtuberosum (sol. tub.). The bottom line labels residues as either strictly conserved (*), highly conserved (:) or weakly conserved (.). Thealignment was generated with the program ClustalX.
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Figure 1: Model of the L36AL protein and of its interaction with tRNA. (A), ribbon representation of the 3-D structure of human RPL36AL(fragment 1-94) modeled by homology with the crystallographic structure of the archaeal counterpart RPL44E of the 60S ribosomal subunitfrom Haloarcula marismortui. The post-translational modifications (including the methylated Q51) [7], and the consensus pattern61Kx(TorV)KKxxL(KorR)xxC72 (numbering of human RPL36AL) of the L44e family of r-proteins are colored pink and cyan, respectively.The 49GG50 dipeptide of the GGQ motif is highlighted in green. A zinc ion represented by a cadmium colored yellow is also shown.Fragment 86-94 corresponding to the nucleotide binding motif 2 (NBD2) common to all eukaryotic RPL7 [29] and RPL36A/RPL44 is shownin wheat. (B), overlaid structures of tRNAPhe (PDB ID 1JGQ) colored grey and of human RPL36AL (blue). The GGQ motif is shown in red,the 3’ terminal CCA trinucleotide of tRNA in pink, and the side chain of Lys-53 in green. (C), NBD2 [29] located in the C-terminal region ofthe eukaryotic RPL7 family (ortholog of the bacterial L30 protein) is conserved in the C-terminal region of the eukaryotic RPL36A/RPL44family. The organisms are: Arabidopsis thaliana (arb. thal.), Drosophila melanogaster (droso.), Saccharomyces cerevisiae (s. cer.), Solanumtuberosum (sol. tub.). The bottom line labels residues as either strictly conserved (*), highly conserved (:) or weakly conserved (.). Thealignment was generated with the program ClustalX.

Mentions: We modeled the 3-D structure of human RPL36AL by homology with the homologous RPL44E of the 50S Hma ribosomal subunit (Fig. 1A), the X-ray crystal structure of which had been determined in a complex with a deacylated tRNA minihelix [8]. We have previously shown that the lysyl residue 53 could be crosslinked to cytidine75ox of tRNAAsp75ox [7], and we have described the post-translational modifications including monomethylated Gln-51 of the 49GGQ51 motif [7, 12] (Fig. 1A). Not surprisingly, the model 3-D structure of RPL36AL was close to that of canine RPL36A observed in a previous study by Chandramouli and co-workers [13]. In this 3-D structure, the methylated GGQ motif (green in Fig. 1A) of RPL36AL occupies a position comparable with that of canine RPL36A and with the corresponding GND motif of the archaeal counterpart L44E from Hma [8] in a loop at one extremity.


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 of the L36AL protein and of its interaction with tRNA. (A), ribbon representation of the 3-D structure of human RPL36AL(fragment 1-94) modeled by homology with the crystallographic structure of the archaeal counterpart RPL44E of the 60S ribosomal subunitfrom Haloarcula marismortui. The post-translational modifications (including the methylated Q51) [7], and the consensus pattern61Kx(TorV)KKxxL(KorR)xxC72 (numbering of human RPL36AL) of the L44e family of r-proteins are colored pink and cyan, respectively.The 49GG50 dipeptide of the GGQ motif is highlighted in green. A zinc ion represented by a cadmium colored yellow is also shown.Fragment 86-94 corresponding to the nucleotide binding motif 2 (NBD2) common to all eukaryotic RPL7 [29] and RPL36A/RPL44 is shownin wheat. (B), overlaid structures of tRNAPhe (PDB ID 1JGQ) colored grey and of human RPL36AL (blue). The GGQ motif is shown in red,the 3’ terminal CCA trinucleotide of tRNA in pink, and the side chain of Lys-53 in green. (C), NBD2 [29] located in the C-terminal region ofthe eukaryotic RPL7 family (ortholog of the bacterial L30 protein) is conserved in the C-terminal region of the eukaryotic RPL36A/RPL44family. The organisms are: Arabidopsis thaliana (arb. thal.), Drosophila melanogaster (droso.), Saccharomyces cerevisiae (s. cer.), Solanumtuberosum (sol. tub.). The bottom line labels residues as either strictly conserved (*), highly conserved (:) or weakly conserved (.). Thealignment was generated with the program ClustalX.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Model of the L36AL protein and of its interaction with tRNA. (A), ribbon representation of the 3-D structure of human RPL36AL(fragment 1-94) modeled by homology with the crystallographic structure of the archaeal counterpart RPL44E of the 60S ribosomal subunitfrom Haloarcula marismortui. The post-translational modifications (including the methylated Q51) [7], and the consensus pattern61Kx(TorV)KKxxL(KorR)xxC72 (numbering of human RPL36AL) of the L44e family of r-proteins are colored pink and cyan, respectively.The 49GG50 dipeptide of the GGQ motif is highlighted in green. A zinc ion represented by a cadmium colored yellow is also shown.Fragment 86-94 corresponding to the nucleotide binding motif 2 (NBD2) common to all eukaryotic RPL7 [29] and RPL36A/RPL44 is shownin wheat. (B), overlaid structures of tRNAPhe (PDB ID 1JGQ) colored grey and of human RPL36AL (blue). The GGQ motif is shown in red,the 3’ terminal CCA trinucleotide of tRNA in pink, and the side chain of Lys-53 in green. (C), NBD2 [29] located in the C-terminal region ofthe eukaryotic RPL7 family (ortholog of the bacterial L30 protein) is conserved in the C-terminal region of the eukaryotic RPL36A/RPL44family. The organisms are: Arabidopsis thaliana (arb. thal.), Drosophila melanogaster (droso.), Saccharomyces cerevisiae (s. cer.), Solanumtuberosum (sol. tub.). The bottom line labels residues as either strictly conserved (*), highly conserved (:) or weakly conserved (.). Thealignment was generated with the program ClustalX.
Mentions: We modeled the 3-D structure of human RPL36AL by homology with the homologous RPL44E of the 50S Hma ribosomal subunit (Fig. 1A), the X-ray crystal structure of which had been determined in a complex with a deacylated tRNA minihelix [8]. We have previously shown that the lysyl residue 53 could be crosslinked to cytidine75ox of tRNAAsp75ox [7], and we have described the post-translational modifications including monomethylated Gln-51 of the 49GGQ51 motif [7, 12] (Fig. 1A). Not surprisingly, the model 3-D structure of RPL36AL was close to that of canine RPL36A observed in a previous study by Chandramouli and co-workers [13]. In this 3-D structure, the methylated GGQ motif (green in Fig. 1A) of RPL36AL occupies a position comparable with that of canine RPL36A and with the corresponding GND motif of the archaeal counterpart L44E from Hma [8] in a loop at one extremity.

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.