National Institutes of Health [GM110588 to M.A.S., D.I.- R.]; California Metabolic Research Foundation; Western University of Health Sciences; U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DEAC02– 76SF00515]; Department of Energy Office of Biological and Environmental Research; National Institutes of Health; National Institute of General Medical Sciences [P41GM103393]. Funding for open access charge: National Institutes of Health; San Diego State University.
Nucleic Acids Research
Nucleosides, Bacteria -- Biosynthesis -- Molecular aspects, Messenger RNA, Adenosine triphosphatase -- Synthesis
The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t6A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteria.
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Luthra, A., Swinehart, W., Bayooz, S., Phan, P., Stec, B., Iwata-Reuyl, D., & Swairjo, M. A. (2018). Structure and mechanism of a bacterial t6A biosynthesis system. Nucleic Acids Research.