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Nucleic Acids Res. 2016 Aug 19;44(14):6971-80. doi: 10.1093/nar/gkw542. Epub 2016 Jun 14.

Structural and functional characterization of KEOPS dimerization by Pcc1 and its role in t6A biosynthesis.

Author information

1
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada.
2
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada.
3
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
4
Department of Medical Biophysics, University of Toronto, Toronto, ON M5S 1L7, Canada.
5
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada Department of Medical Biophysics, University of Toronto, Toronto, ON M5S 1L7, Canada.
6
Cornell University, Department of Chemistry and Chemical Biology, NE-CAT, Building 436E, Advanced Photon Source, 9700 S. Cass Avenue, Argonne, IL 60439, USA.
7
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada sicheri@lunenfeld.ca.

Abstract

KEOPS is an ancient protein complex required for the biosynthesis of N6-threonylcarbamoyladenosine (t(6)A), a universally conserved tRNA modification found on all ANN-codon recognizing tRNAs. KEOPS consist minimally of four essential subunits, namely the proteins Kae1, Bud32, Cgi121 and Pcc1, with yeast possessing the fifth essential subunit Gon7. Bud32, Cgi121, Pcc1 and Gon7 appear to have evolved to regulate the central t(6)A biosynthesis function of Kae1, but their precise function and mechanism of action remains unclear. Pcc1, in particular, binds directly to Kae1 and by virtue of its ability to form dimers in solution and in crystals, Pcc1 was inferred to function as a dimerization module for Kae1 and therefore KEOPS. We now present a 3.4 Å crystal structure of a dimeric Kae1-Pcc1 complex providing direct evidence that Pcc1 can bind and dimerize Kae1. Further biophysical analysis of a complete archaeal KEOPS complex reveals that Pcc1 facilitates KEOPS dimerization in vitro Interestingly, while Pcc1-mediated dimerization of KEOPS is required to support the growth of yeast, it is dispensable for t(6)A biosynthesis by archaeal KEOPS in vitro, raising the question of how precisely Pcc1-mediated dimerization impacts cellular biology.

PMID:
27302132
PMCID:
PMC5001605
DOI:
10.1093/nar/gkw542
[Indexed for MEDLINE]
Free PMC Article

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