Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa.
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 tRNAMet(CAU)and tRNATrp(CCA)are substrates for Cm formation, tRNAGln(UUG), tRNAPro(UGG), tRNAPro(CGG)and tRNAHis(GUG)for Um, and tRNAPro(GGG)for Am. tRNASer(UGA), 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 tRNAPro(GGG)and Um in tRNAGln(UUG)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 H2O2exposure, 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.
Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.|Singapore-MIT Alliance for Research and Technology, Singapore.
Juthamas
J
Jaroensuk
Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand.
Sopapan
S
Atichartpongkul
Singapore-MIT Alliance for Research and Technology, Singapore.
Yok Hian
YH
Chionh
School of Biological Sciences, Nanyang Technological University, Singapore.
Yee Hwa
YH
Wong
NTU Institute of Structural Biology, Nanyang Technological University, Singapore.
Chong Wai
CW
Liew
Singapore-MIT Alliance for Research and Technology, Singapore.
Megan E
ME
McBee
Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.
Narumon
N
Thongdee
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Erin G
EG
Prestwich
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Michael S
MS
DeMott
Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand.|Department of Biotechnology, Faculty of Sciences, Mahidol University, Bangkok, Thailand.|Center of Excellence on Environmental Health and Toxicology (EHT), Bangkok, Thailand.
Skorn
S
Mongkolsuk
Singapore-MIT Alliance for Research and Technology, Singapore pcdedon@mit.edu.|Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Peter C
PC
Dedon
School of Biological Sciences, Nanyang Technological University, Singapore julien@ntu.edu.sg.|NTU Institute of Structural Biology, Nanyang Technological University, Singapore.|UPMC UMRS CR7 - CNRS ERL 8255-INSERM U1135 Centre d' Immunologie et des Maladies Infectieuses, Paris, France.
Julien
J
Lescar
Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand mayuree@cri.or.th.|Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand.|Center of Excellence on Environmental Health and Toxicology (EHT), Bangkok, Thailand.
Mayuree
0000-0001-5684-9001
M
Fuangthong
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008745:Methylation; D008958:Models, Molecular; D018384:Oxidative Stress; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology*; D012329:RNA, Bacterial / Q000737:chemistry; D012343:RNA, Transfer / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000502:physiology
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012343:RNA, Transfer; D006861:Hydrogen Peroxide; D012359:tRNA Methyltransferases
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