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Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Perrochia L, Guetta D, Hecker A, Forterre P, Basta T - Nucleic Acids Res. (2013)

Bottom Line: We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex.Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA.Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.

ABSTRACT
N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

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ATPase activity of wild-type and mutant KEOPS proteins from P. abyssi. KEOPS subcomplexes were incubated with α-P32 ATP in reaction buffer either alone or in combination with other proteins. Produced radiolabeled (α-P32)-ADP was separated by TLC and visualized by phosphorimaging. When indicated, Ec_tRNALys produced in overexpressing E. coli strain was added to the reaction mixture. In negative controls, indicated by a minus sign, the proteins were omitted in the reaction mixture. (A): ATPase activity of binary complexes. (B): ATPase activity of ternary complex Kae1–Bud32–Cgi121. (C): The ATPase activity was measured for reconstituted KEOPS complexes containing point mutations in Kae1 and Bud32 indicated at the bottom. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.
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gkt720-F3: ATPase activity of wild-type and mutant KEOPS proteins from P. abyssi. KEOPS subcomplexes were incubated with α-P32 ATP in reaction buffer either alone or in combination with other proteins. Produced radiolabeled (α-P32)-ADP was separated by TLC and visualized by phosphorimaging. When indicated, Ec_tRNALys produced in overexpressing E. coli strain was added to the reaction mixture. In negative controls, indicated by a minus sign, the proteins were omitted in the reaction mixture. (A): ATPase activity of binary complexes. (B): ATPase activity of ternary complex Kae1–Bud32–Cgi121. (C): The ATPase activity was measured for reconstituted KEOPS complexes containing point mutations in Kae1 and Bud32 indicated at the bottom. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.

Mentions: As shown in Figure 3A, binary complex Kae1–Bud32 exhibited a significant ATPase activity in the presence or absence of tRNA, whereas the two other binary complexes Pcc1–Kae1 and Bud32–Cgi121 did not. This indicated that Kae1 and/or Bud32 were responsible for the ATPase activity of PaKEOPS, but only when the two proteins interact with each other. Interestingly, whereas the ATPase activity of the whole PaKEOPS complex is stimulated by the presence of tRNA, the ATPase activity of the Kae1–Bud32 complex is independent of tRNA, suggesting that Pcc1 and/or Cgi121 were required for the response to tRNA. To test this hypothesis, we compared the ATPase activity of Kae1–Bud32–Cgi121 complex with that of the mixture of the Pcc1–Kae1 and Kae1–Bud32 binary complexes (Figure 3B). In both cases, we recorded a significant ATPase activity that was independent of tRNA. However, when Kae1–Bud32–Cgi121 was combined with Pcc1–Kae1, or Cgi121 was added to the Pcc1–Kae1 and Kae1–Bud32 assay, we observed the wild-type tRNA-stimulated ATPase activity. Taken together, these data indicate that the simultaneous presence of both Pcc1 and Cgi121 is required for the tRNA-stimulated ATPase activity of PaKEOPS.Figure 3.


Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Perrochia L, Guetta D, Hecker A, Forterre P, Basta T - Nucleic Acids Res. (2013)

ATPase activity of wild-type and mutant KEOPS proteins from P. abyssi. KEOPS subcomplexes were incubated with α-P32 ATP in reaction buffer either alone or in combination with other proteins. Produced radiolabeled (α-P32)-ADP was separated by TLC and visualized by phosphorimaging. When indicated, Ec_tRNALys produced in overexpressing E. coli strain was added to the reaction mixture. In negative controls, indicated by a minus sign, the proteins were omitted in the reaction mixture. (A): ATPase activity of binary complexes. (B): ATPase activity of ternary complex Kae1–Bud32–Cgi121. (C): The ATPase activity was measured for reconstituted KEOPS complexes containing point mutations in Kae1 and Bud32 indicated at the bottom. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814370&req=5

gkt720-F3: ATPase activity of wild-type and mutant KEOPS proteins from P. abyssi. KEOPS subcomplexes were incubated with α-P32 ATP in reaction buffer either alone or in combination with other proteins. Produced radiolabeled (α-P32)-ADP was separated by TLC and visualized by phosphorimaging. When indicated, Ec_tRNALys produced in overexpressing E. coli strain was added to the reaction mixture. In negative controls, indicated by a minus sign, the proteins were omitted in the reaction mixture. (A): ATPase activity of binary complexes. (B): ATPase activity of ternary complex Kae1–Bud32–Cgi121. (C): The ATPase activity was measured for reconstituted KEOPS complexes containing point mutations in Kae1 and Bud32 indicated at the bottom. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.
Mentions: As shown in Figure 3A, binary complex Kae1–Bud32 exhibited a significant ATPase activity in the presence or absence of tRNA, whereas the two other binary complexes Pcc1–Kae1 and Bud32–Cgi121 did not. This indicated that Kae1 and/or Bud32 were responsible for the ATPase activity of PaKEOPS, but only when the two proteins interact with each other. Interestingly, whereas the ATPase activity of the whole PaKEOPS complex is stimulated by the presence of tRNA, the ATPase activity of the Kae1–Bud32 complex is independent of tRNA, suggesting that Pcc1 and/or Cgi121 were required for the response to tRNA. To test this hypothesis, we compared the ATPase activity of Kae1–Bud32–Cgi121 complex with that of the mixture of the Pcc1–Kae1 and Kae1–Bud32 binary complexes (Figure 3B). In both cases, we recorded a significant ATPase activity that was independent of tRNA. However, when Kae1–Bud32–Cgi121 was combined with Pcc1–Kae1, or Cgi121 was added to the Pcc1–Kae1 and Kae1–Bud32 assay, we observed the wild-type tRNA-stimulated ATPase activity. Taken together, these data indicate that the simultaneous presence of both Pcc1 and Cgi121 is required for the tRNA-stimulated ATPase activity of PaKEOPS.Figure 3.

Bottom Line: We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex.Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA.Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.

ABSTRACT
N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

Show MeSH
Related in: MedlinePlus