Published on Dec. 15, 2016 in Nucleic Acids Res volume 44 (22).
PubMed ID: 27683218
Abstract:
Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNA<sup>Met(CAU)</sup> and tRNA<sup>Trp(CCA)</sup> are substrates for Cm formation, tRNA<sup>Gln(UUG)</sup>, tRNA<sup>Pro(UGG)</sup>, tRNA<sup>Pro(CGG)</sup> and tRNA<sup>His(GUG)</sup> for Um, and tRNA<sup>Pro(GGG)</sup> for Am. tRNA<sup>Ser(UGA)</sup>, previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNA<sup>Pro(GGG)</sup> and Um in tRNA<sup>Gln(UUG)</sup> by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H<sub>2</sub>O<sub>2</sub> exposure, with reduced expression of oxyR-recG, katB-ankB, and katE These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.