tRNA epitranscriptomics and biased codon are linked to proteome expression in

Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites.Singapore. National Research FoundationSingapore-MIT Alliance (Graduate Fellowship

Backbone resonance assignment for the full length tRNA-(N1G37) methyltransferase of Pseudomonas aeruginosa

Li, Y. et al. "Backbone resonance assignment for the full length tRNA-(N1G37) methyltransferase of Pseudomonas aeruginosa." Biomolecular NMR Assignments 13, 2 (June 2019): 327–332 © 2019 Springer Natur

Backbone resonance assignment for the N-terminal region of bacterial tRNA-(N1G37) methyltransferase

Bacterial tRNA (guanine³⁷-N¹)-methyltransferase (TrmD) is an important antibacterial target due to its essential role in translation. TrmD has two domains connected with a flexible linker. The N-terminal domain (NTD) of TrmD contains the S-adenosyl-l-methionine (SAM) cofactor binding site and the C-terminal domain is critical for tRNA binding. Here we report the backbone NMR resonance assignments for NTD of Pseudomonas aeruginosa TrmD. Its secondary structure was determined based on the assigned resonances. Relaxation analysis revealed that NTD existed as dimers in solution. NTD also exhibited thermal stability in solution. Its interactions with SAM and other compounds suggest it can be used for evaluating SAM competitive inhibitors by NMR

Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-N¹)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism

Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N1G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors

Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N1G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-l-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAM-noncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)National Research Foundation (NRF)Singapore-MIT Alliance for Research and Technology (SMART)Published versionThis research was supported by the National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology (SMART) Infectious Diseases and Antimicrobial Resistance Interdisciplinary Research Groups; SMART Innovation Centre Grant ING137070-BIO to P.D. and J.L.; the Biomedical Sciences Institutes (BMSI), Agency for Science, Technology, and Research (A*STAR), Singapore; and AcRF Grants Tier1 RG154/14 and MOE2015- T2-2-075 to J.L