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Title: Conformational communication mediates the reset step in t6A biosynthesis

Abstract

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

Authors:
 [1];  [1];  [2];  [1];  [1];  [1];  [3];  [1];  [2];  [4]
  1. Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA
  2. Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA
  3. Schrödinger, 10201 Wateridge Cir Suite 220, San Diego, CA 92121, USA
  4. Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA, The Viral Information Institute, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA
Publication Date:
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1530481
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research Journal Volume: 47 Journal Issue: 12; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Luthra, Amit, Paranagama, Naduni, Swinehart, William, Bayooz, Susan, Phan, Phuc, Quach, Vanessa, Schiffer, Jamie M., Stec, Boguslaw, Iwata-Reuyl, Dirk, and Swairjo, Manal A. Conformational communication mediates the reset step in t6A biosynthesis. United Kingdom: N. p., 2019. Web. doi:10.1093/nar/gkz439.
Luthra, Amit, Paranagama, Naduni, Swinehart, William, Bayooz, Susan, Phan, Phuc, Quach, Vanessa, Schiffer, Jamie M., Stec, Boguslaw, Iwata-Reuyl, Dirk, & Swairjo, Manal A. Conformational communication mediates the reset step in t6A biosynthesis. United Kingdom. doi:10.1093/nar/gkz439.
Luthra, Amit, Paranagama, Naduni, Swinehart, William, Bayooz, Susan, Phan, Phuc, Quach, Vanessa, Schiffer, Jamie M., Stec, Boguslaw, Iwata-Reuyl, Dirk, and Swairjo, Manal A. Wed . "Conformational communication mediates the reset step in t6A biosynthesis". United Kingdom. doi:10.1093/nar/gkz439.
@article{osti_1530481,
title = {Conformational communication mediates the reset step in t6A biosynthesis},
author = {Luthra, Amit and Paranagama, Naduni and Swinehart, William and Bayooz, Susan and Phan, Phuc and Quach, Vanessa and Schiffer, Jamie M. and Stec, Boguslaw and Iwata-Reuyl, Dirk and Swairjo, Manal A.},
abstractNote = {Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.},
doi = {10.1093/nar/gkz439},
journal = {Nucleic Acids Research},
number = 12,
volume = 47,
place = {United Kingdom},
year = {2019},
month = {5}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/nar/gkz439

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