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

Abstract

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, tRNA^LeuCAA, tRNA^PheGAA, and tRNA^SerCGA from M. tb, confirming the role of Arg residue in tRNA binding.