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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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Impact of point mutations of Kae1 and Bud32 on the in vitro biosynthesis of t6A modification by KEOPS from P. abyssi. Whole KEOPS complexes were reconstituted by mixing two binary subcomplexes carrying point mutations. The reconstituted complexes were incubated with Sua5 from P. abyssi, ATP, bicarbonate, C14l-threonine and substrate tRNA. The reaction mixtures were precipitated with TCA, and the radioactivity levels in the precipitates were measured by scintillation counting. The counts were converted into amount of incorporated C14l-threonine into tRNA using a standard calibration curve. The negative control, which is designated with a minus sign, corresponds to the total assay mixture without proteins.
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gkt720-F4: Impact of point mutations of Kae1 and Bud32 on the in vitro biosynthesis of t6A modification by KEOPS from P. abyssi. Whole KEOPS complexes were reconstituted by mixing two binary subcomplexes carrying point mutations. The reconstituted complexes were incubated with Sua5 from P. abyssi, ATP, bicarbonate, C14l-threonine and substrate tRNA. The reaction mixtures were precipitated with TCA, and the radioactivity levels in the precipitates were measured by scintillation counting. The counts were converted into amount of incorporated C14l-threonine into tRNA using a standard calibration curve. The negative control, which is designated with a minus sign, corresponds to the total assay mixture without proteins.

Mentions: Next, whole PaKEOPS complexes that contained either Kae1D159A or Bud32D127R were reconstituted and their ATPase activity was measured in presence or absence of tRNA. We found that PaKEOPS (Kae1D159A) behaved in the same manner as the wild-type PaKEOPS, whereas PaKEOPS (Bud32D127R) totally lost the ATPase activity (Figure 3C). This indicated that Bud32 is the subunit responsible for the ATPase activity of PaKEOPS. Moreover, PaKEOPS (Bud32D127R) was inactive when assayed for the t6A synthetic activity, indicating that the ATPase activity of Bud32 is most likely required for the biosynthesis of t6A (Figure 4).Figure 4.


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)

Impact of point mutations of Kae1 and Bud32 on the in vitro biosynthesis of t6A modification by KEOPS from P. abyssi. Whole KEOPS complexes were reconstituted by mixing two binary subcomplexes carrying point mutations. The reconstituted complexes were incubated with Sua5 from P. abyssi, ATP, bicarbonate, C14l-threonine and substrate tRNA. The reaction mixtures were precipitated with TCA, and the radioactivity levels in the precipitates were measured by scintillation counting. The counts were converted into amount of incorporated C14l-threonine into tRNA using a standard calibration curve. The negative control, which is designated with a minus sign, corresponds to the total assay mixture without proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt720-F4: Impact of point mutations of Kae1 and Bud32 on the in vitro biosynthesis of t6A modification by KEOPS from P. abyssi. Whole KEOPS complexes were reconstituted by mixing two binary subcomplexes carrying point mutations. The reconstituted complexes were incubated with Sua5 from P. abyssi, ATP, bicarbonate, C14l-threonine and substrate tRNA. The reaction mixtures were precipitated with TCA, and the radioactivity levels in the precipitates were measured by scintillation counting. The counts were converted into amount of incorporated C14l-threonine into tRNA using a standard calibration curve. The negative control, which is designated with a minus sign, corresponds to the total assay mixture without proteins.
Mentions: Next, whole PaKEOPS complexes that contained either Kae1D159A or Bud32D127R were reconstituted and their ATPase activity was measured in presence or absence of tRNA. We found that PaKEOPS (Kae1D159A) behaved in the same manner as the wild-type PaKEOPS, whereas PaKEOPS (Bud32D127R) totally lost the ATPase activity (Figure 3C). This indicated that Bud32 is the subunit responsible for the ATPase activity of PaKEOPS. Moreover, PaKEOPS (Bud32D127R) was inactive when assayed for the t6A synthetic activity, indicating that the ATPase activity of Bud32 is most likely required for the biosynthesis of t6A (Figure 4).Figure 4.

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