Year: 2017
Type: article
Abstract: 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.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkx1300
Institutions: San Diego State University, Portland State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,992
Awards: G1229526393, G1286251239, G1804828992, G1831385526, G2371548206, G303061748, G315621713, G3344126041, G3488436650, G3629260272, G4581610328, RNA modification: Mechanism and links to other metabolic pathways, G495301720, G498139845, G5570207115, G6550344761, G7283024673, G8382000481, A Synchrotron Radiation Structural Biology Resource, G8925327636, G911061179, G969889393
OpenAlex ID: W2782363424