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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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Binding of tRNA by PaKEOPS subcomplexes and individual proteins. Individual PaKEOPS subunits and subcomplexes were mixed with 10 nM of radiolabeled (P32) tRNA at room temperature and the mixture was separated by native PAGE (see ‘Materials and Methods’ section). The radioactive bands corresponding to unbound tRNA (at the bottom of each gel) and the nucleoprotein complexes were recorded by phosphorimaging. Protein concentrations used are indicated in µM at the top of each panel. In the downright panel, fixed concentration of the Kae1–Bud32 complex (0.5 µM) was titrated with increasing concentrations of Cgi121, which are indicated for each sample at the top of the gel. Minus sign stands for the control sample to which proteins were not added. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.
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gkt720-F7: Binding of tRNA by PaKEOPS subcomplexes and individual proteins. Individual PaKEOPS subunits and subcomplexes were mixed with 10 nM of radiolabeled (P32) tRNA at room temperature and the mixture was separated by native PAGE (see ‘Materials and Methods’ section). The radioactive bands corresponding to unbound tRNA (at the bottom of each gel) and the nucleoprotein complexes were recorded by phosphorimaging. Protein concentrations used are indicated in µM at the top of each panel. In the downright panel, fixed concentration of the Kae1–Bud32 complex (0.5 µM) was titrated with increasing concentrations of Cgi121, which are indicated for each sample at the top of the gel. Minus sign stands for the control sample to which proteins were not added. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.

Mentions: We previously demonstrated that the PaKEOPS complex binds tightly to tRNA; however, the contribution of each subunit in the binding process was unknown (23). We used electrophoretic mobility shift analysis (EMSA) experiments to directly measure binding of different PaKEOPS subunits to radioactively labeled substrate tRNA and compare their binding profile relative to the whole PaKEOPS complex (Figure 7). Formation of a discrete shifted band was visible already at 10 nM of PaKEOPS (corresponding to molar ratio protein:tRNA of 1:1) with apparent Kd value of 100–500 nM (corresponding to 50% of bound tRNA). Using Kae1 alone, we observed formation of diffuse bands of retarded tRNA starting from 1 µM concentration of protein, and almost the entire probe was retarded at 2 µM of Kae1.Figure 7.


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)

Binding of tRNA by PaKEOPS subcomplexes and individual proteins. Individual PaKEOPS subunits and subcomplexes were mixed with 10 nM of radiolabeled (P32) tRNA at room temperature and the mixture was separated by native PAGE (see ‘Materials and Methods’ section). The radioactive bands corresponding to unbound tRNA (at the bottom of each gel) and the nucleoprotein complexes were recorded by phosphorimaging. Protein concentrations used are indicated in µM at the top of each panel. In the downright panel, fixed concentration of the Kae1–Bud32 complex (0.5 µM) was titrated with increasing concentrations of Cgi121, which are indicated for each sample at the top of the gel. Minus sign stands for the control sample to which proteins were not added. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt720-F7: Binding of tRNA by PaKEOPS subcomplexes and individual proteins. Individual PaKEOPS subunits and subcomplexes were mixed with 10 nM of radiolabeled (P32) tRNA at room temperature and the mixture was separated by native PAGE (see ‘Materials and Methods’ section). The radioactive bands corresponding to unbound tRNA (at the bottom of each gel) and the nucleoprotein complexes were recorded by phosphorimaging. Protein concentrations used are indicated in µM at the top of each panel. In the downright panel, fixed concentration of the Kae1–Bud32 complex (0.5 µM) was titrated with increasing concentrations of Cgi121, which are indicated for each sample at the top of the gel. Minus sign stands for the control sample to which proteins were not added. Single letter designations are as follows: P, Pcc1; K, Kae1; B, Bud32 and C, Cgi121.
Mentions: We previously demonstrated that the PaKEOPS complex binds tightly to tRNA; however, the contribution of each subunit in the binding process was unknown (23). We used electrophoretic mobility shift analysis (EMSA) experiments to directly measure binding of different PaKEOPS subunits to radioactively labeled substrate tRNA and compare their binding profile relative to the whole PaKEOPS complex (Figure 7). Formation of a discrete shifted band was visible already at 10 nM of PaKEOPS (corresponding to molar ratio protein:tRNA of 1:1) with apparent Kd value of 100–500 nM (corresponding to 50% of bound tRNA). Using Kae1 alone, we observed formation of diffuse bands of retarded tRNA starting from 1 µM concentration of protein, and almost the entire probe was retarded at 2 µM of Kae1.Figure 7.

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