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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.


Plot of the molar fraction of crosslinking as a function of pH. Two data sets from two separate experiments of Fig. (5B) were used to obtain this graph. The data were fitted with Mathematica to the function inscribed in the figure with the following features : the dashedlines defines the 95% confidence intervals for the prediction of a single value, whereas the continous lines and the gray-shaded region define the 95% confidence intervals for the prediction of the average curve with pK = 6.9 ± 0.2.
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Figure 9: Plot of the molar fraction of crosslinking as a function of pH. Two data sets from two separate experiments of Fig. (5B) were used to obtain this graph. The data were fitted with Mathematica to the function inscribed in the figure with the following features : the dashedlines defines the 95% confidence intervals for the prediction of a single value, whereas the continous lines and the gray-shaded region define the 95% confidence intervals for the prediction of the average curve with pK = 6.9 ± 0.2.

Mentions: Assuming that the maximal yield of crosslinking in the binary RPL36AL-tRNAox complex “a” was obtained at pH 8.0 and thus considered as 100%, the yields of crosslinking for the “a” band from Fig. (5B) (lanes 1 to 6) were 10% at pH 5.0, 23% at pH 6.0, 50% at pH 7.0, 67% at pH 7.5, 100% at pH 8.0 and 90% at pH 9.0. These data were then used to plot the crosslinking yield versus increasing pH values. As shown in Fig. (9), the crosslinking yield is proportional to the pH, i.e. to the mole fraction of a basic group with a pKapp of about 6.9 describing the dissociation of the protonated form of the reactive ε-amino group of Lys-53. This value of pKapp is 3 to 3.5 units lower than is normally found for an ε-amino group of a lysyl residue in a protein, and it is likely that this abnormally low pKapp accounts for the reactivity of Lys-53 through the Schiff base formation leading to crosslinking. In conclusion, the abnormally low pKapp of the ε-amino group of Lys-53 would result in a greater mole fraction in the reactive, unprotonated form at neutral pH values, and consequently a greater crosslinking yield. Since, in general, unusually reactive side chains are also those required for biological activity, so that pH dependence of activity can be correlated with pH dependence of the modification reaction, it is most probable that the ε-amino group of Lys-53 with a pKapp of 6.9 might be of functional importance for the biological activity of human 80S ribosome. Even though at pH near 5.0 the human 80S ribosomes can be inactivated due to precipitation, the mole fraction in the reactive, unprotonated form of the crosslinked residues and consequently the crosslinking yields would not be affected.


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)

Plot of the molar fraction of crosslinking as a function of pH. Two data sets from two separate experiments of Fig. (5B) were used to obtain this graph. The data were fitted with Mathematica to the function inscribed in the figure with the following features : the dashedlines defines the 95% confidence intervals for the prediction of a single value, whereas the continous lines and the gray-shaded region define the 95% confidence intervals for the prediction of the average curve with pK = 6.9 ± 0.2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Plot of the molar fraction of crosslinking as a function of pH. Two data sets from two separate experiments of Fig. (5B) were used to obtain this graph. The data were fitted with Mathematica to the function inscribed in the figure with the following features : the dashedlines defines the 95% confidence intervals for the prediction of a single value, whereas the continous lines and the gray-shaded region define the 95% confidence intervals for the prediction of the average curve with pK = 6.9 ± 0.2.
Mentions: Assuming that the maximal yield of crosslinking in the binary RPL36AL-tRNAox complex “a” was obtained at pH 8.0 and thus considered as 100%, the yields of crosslinking for the “a” band from Fig. (5B) (lanes 1 to 6) were 10% at pH 5.0, 23% at pH 6.0, 50% at pH 7.0, 67% at pH 7.5, 100% at pH 8.0 and 90% at pH 9.0. These data were then used to plot the crosslinking yield versus increasing pH values. As shown in Fig. (9), the crosslinking yield is proportional to the pH, i.e. to the mole fraction of a basic group with a pKapp of about 6.9 describing the dissociation of the protonated form of the reactive ε-amino group of Lys-53. This value of pKapp is 3 to 3.5 units lower than is normally found for an ε-amino group of a lysyl residue in a protein, and it is likely that this abnormally low pKapp accounts for the reactivity of Lys-53 through the Schiff base formation leading to crosslinking. In conclusion, the abnormally low pKapp of the ε-amino group of Lys-53 would result in a greater mole fraction in the reactive, unprotonated form at neutral pH values, and consequently a greater crosslinking yield. Since, in general, unusually reactive side chains are also those required for biological activity, so that pH dependence of activity can be correlated with pH dependence of the modification reaction, it is most probable that the ε-amino group of Lys-53 with a pKapp of 6.9 might be of functional importance for the biological activity of human 80S ribosome. Even though at pH near 5.0 the human 80S ribosomes can be inactivated due to precipitation, the mole fraction in the reactive, unprotonated form of the crosslinked residues and consequently the crosslinking yields would not be affected.

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.