Conformational communication mediates the reset step in t6A biosynthesis

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 thre...

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Published in:	Nucleic acids research Vol. 47; no. 12; pp. 6551 - 6567
Main Authors:	Luthra, Amit, Paranagama, Naduni, Swinehart, William, Bayooz, Susan, Phan, Phuc, Quach, Vanessa, Schiffer, Jamie M, Stec, Boguslaw, Iwata-Reuyl, Dirk, Swairjo, Manal A
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 09-07-2019
Subjects:	Adenosine - analogs & derivatives Adenosine - biosynthesis Adenosine Triphosphatases - genetics Amino Acid Motifs Bacterial Proteins - chemistry Bacterial Proteins - genetics Models, Molecular Mutagenesis Protein Conformation RNA, Transfer - chemistry RNA, Transfer - metabolism Structural Biology Thermotoga maritima
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Summary:	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.
Bibliography:	USDOE Office of Science (SC), Basic Energy Sciences (BES) AC02-76SF00515
ISSN:	0305-1048 1362-4962
DOI:	10.1093/nar/gkz439