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

Abstract

KEOPS is an ancient protein complex required for the biosynthesis of N6-threonylcarbamoyladenosine (t6A), 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 t6A 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 t6A biosynthesis by archaeal KEOPS in vitro, raising the question of how precisely Pcc1-mediated dimerization impacts cellular biology.

Authors:
 [1];  [2];  [1];  [3];  [3];  [2];  [4];  [1];  [1]
  1. Lunenfeld-Tanenbaum Research Inst., Toronto, ON (Canada); Univ. of Toronto, ON (Canada)
  2. McMaster Univ., Hamilton, ON (Canada)
  3. Univ. of Toronto, ON (Canada)
  4. Cornell Univ., Argonne, IL (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
Canadian Inst. of Health Research (CIHR) Foundation; National Inst. of Health; NIH-ORIP HEI
OSTI Identifier:
1328017
Grant/Contract Number:  
AC02-06CH11357; FDN 143277; FDN 143343; P41 GM103403; S10 RR029205
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 44; Journal Issue: 14; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Wan, Leo C. K., Pillon, Monica C., Thevakumaran, Neroshan, Sun, Yulong, Chakrabartty, Avi, Guarné, Alba, Kurinov, Igor, Durocher, Daniel, and Sicheri, Frank. Structural and functional characterization of KEOPS dimerization by Pcc1 and its role in t6A biosynthesis. United States: N. p., 2016. Web. doi:10.1093/nar/gkw542.
Wan, Leo C. K., Pillon, Monica C., Thevakumaran, Neroshan, Sun, Yulong, Chakrabartty, Avi, Guarné, Alba, Kurinov, Igor, Durocher, Daniel, & Sicheri, Frank. Structural and functional characterization of KEOPS dimerization by Pcc1 and its role in t6A biosynthesis. United States. https://doi.org/10.1093/nar/gkw542
Wan, Leo C. K., Pillon, Monica C., Thevakumaran, Neroshan, Sun, Yulong, Chakrabartty, Avi, Guarné, Alba, Kurinov, Igor, Durocher, Daniel, and Sicheri, Frank. Tue . "Structural and functional characterization of KEOPS dimerization by Pcc1 and its role in t6A biosynthesis". United States. https://doi.org/10.1093/nar/gkw542. https://www.osti.gov/servlets/purl/1328017.
@article{osti_1328017,
title = {Structural and functional characterization of KEOPS dimerization by Pcc1 and its role in t6A biosynthesis},
author = {Wan, Leo C. K. and Pillon, Monica C. and Thevakumaran, Neroshan and Sun, Yulong and Chakrabartty, Avi and Guarné, Alba and Kurinov, Igor and Durocher, Daniel and Sicheri, Frank},
abstractNote = {KEOPS is an ancient protein complex required for the biosynthesis of N6-threonylcarbamoyladenosine (t6A), 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 t6A 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 t6A biosynthesis by archaeal KEOPS in vitro, raising the question of how precisely Pcc1-mediated dimerization impacts cellular biology.},
doi = {10.1093/nar/gkw542},
journal = {Nucleic Acids Research},
number = 14,
volume = 44,
place = {United States},
year = {Tue Jun 14 00:00:00 EDT 2016},
month = {Tue Jun 14 00:00:00 EDT 2016}
}

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Works referencing / citing this record:

Defects in t6A tRNA modification due to GON7 and YRDC mutations lead to Galloway-Mowat syndrome
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Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA
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