Arginine to glutamine mutation in the substrate binding region impaired the isopentenyl activity of Mycobacterium tuberculosis MiaA

Document Type : Original article


1 School of Biotechnology, Gautam Buddha University, Gautam Budh Nagar, Greater Noida, Uttar Pradesh, India

2 School of Sciences, Indira Gandhi National Open University, Maidan Garhi, New Delhi, India


tRNAs act as adaptors during protein synthesis and are chemically modified post-transcriptionally for their structural stability as well as accuracy of the translation. Hypomodifications of tRNAs are known to cause various human diseases, including cancer. Studies in bacteria and yeasts showed that levels of tRNA modifications vary under different stress conditions, enabling the organism to modulate gene expression for survival. Isopentelylation of the base 37 (i6A37) in the anticodon stem-loop by tRNA isopentenyltransferase (MiaA) is well-conserved modification present in prokaryotes and eukaryotes. i6A37 modification increases both the speed and fidelity of translation. A homozygous p.Arg323Gln mutation in the tRNA binding region of tRNA isopentenyltransferase reduced i6A37 levels in humans, affecting mitochondrial translation and thereby causing neurodevelopmental disorder. In this study, we mutated the Arg residue at the conserved position to Gln in Mycobacterium tuberculosis (M. tb) MiaA and analyzed the i6A modification activity of the enzyme on its target tRNAs. We found that p.Arg274Gln mutant MiaA could not modify the target tRNAs, tRNALeuCAA, tRNAPheGAA, and tRNASerCGA from M. tb, confirming the role of Arg residue in tRNA binding.


  1. Torres AG, Batlle E, Ribas de Pouplana L. Role of tRNA modifications in human diseases. Trends Mol Med 2014;20:306-314.
  2. Schweizer U, Bohleber S, Fradejas-Villar N. The modified base isopentenyladenosine and its derivatives in tRNA. RNA Biol 2017;14:1197-1208.
  3. Khalique A, Mattijssen S, Haddad AF, Maraia RJ. Targeting of mitochondrial and cytosolic substrates of tRNA isopentenyltransferases: 2 selection of differential tRNA-i6A37 identity subsets. PLoS Genet 2020;16:e1008330.
  4. Xie W, Zhou C, Huang RH. Structure of tRNA dimethylallyltransferase: RNA modification through a channel. J Mol Biol 2007;367:872-881.
  5. Zhou C, Huang RH. Crystallographic snapshots of eukaryotic dimethylallyltransferase acting on tRNA: insight into tRNA recognition and reaction mechanism. Proc Natl Acad Sci USA 2008;105:16142-16147.
  6. Yarham JW, Lamichhane TN, Pyle A, Mattijssen S, Baruffini E, Bruni F, Donnini C, Vassilev A, He L, Blakely EL, Griffin H, Santibanez-Koref M, Bindoff LA, Ferrero I, Chinnery PF, McFarland R, Maraia RJ, Taylor RW. Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA. PLoS Genet 2014;10:e1004424.
  7. Soman S, Ram S. MiaA (Rv2727c) mediated tRNA isopentenylation of Mycobacterium tuberculosis H37Rv. Mol Biol Res Commun 2022;11:97-104.
  8. Goude R, Parish T. Electroporation of Mycobacteria. In: Parish T, Brown AC, editors. Mycobacteria Protocols: Second Edition. Totowa, NJ: Humana Press; 2009. pp. 203-215.
  9. Chambers AE, Richardson AE, Read DF, Waller TJ, Bernstein DA, Smaldino PJ. An in vitro assay to detect tRNA-isopentenyl transferase activity. J Vis Exp 2018;8:58100.
  10. Thompson KM, Gottesman S. The MiaA tRNA modification enzyme is necessary for robust RpoS expression in Escherichia coli. J Bacteriol 2014;196:754-761.
  11. Urbonavicius J, Durand JMB, Björk GR. Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri. J Bacteriol 2002;184:5348-5357.
  12. Fleming BA, Blango MG, Rousek AA, Kincannon WM, Tran A, Lewis AJ, Russell CW, Zhou Q, Baird LM, Barber AE, Brannon JR, Beebout CJ, Bandarian V, Hadjifrangiskou M, Howard MT, Mulvey M. A tRNA modifying enzyme as a tunable regulatory nexus for bacterial stress responses and virulence. Nucleic Acids Res 2022;50:7570-7590.
  13. Global Tuberculosis Report, WHO 2021. In: [Internet]. 14 Oct 2021 [cited 20 Apr 2022]. Available:
  14. Eoh H, Brennan PJ, Crick DC. The Mycobacterium tuberculosis MEP (2C-methyl-d-erythritol 4-phosphate) pathway as a new drug target. Tuberculosis 2009;89:1-11.