Fig. 3. MenT1 inhibits translation by modifying tRNAs in vitro.

A Total tRNA from E. coli was pre-incubated with MenT1 in vitro and subsequently used in a cell-free translation assay. Samples were separated on 4–20% SDS-PAGE gel and expression levels of both GFP and WaaF model substrates were determined using anti-GFP antibody and autoradiography, respectively. B tRNA modification by MenT1 was analysed by incubating 1 µg of total RNA from M. smegmatis, M. tuberculosis, or E. coli or 100 ng of purified E. coli tRNA with MenT1 (5 µM) in the presence of [α−³²P] labelled NTPs at 37 °C for 2 h. Samples were separated on 6% Urea-PAGE gel and revealed by autoradiography. C Total RNA from M. smegmatis was incubated with MenT1 (5 µM) in the presence of [α−³²P] labelled ATP, UTP, GTP, or CTP at 37 °C for 2 h. Representative results of triplicate experiments are shown. Source data are provided as a Source data file. D tRNA 3′-ends modified by MenT1 were mapped by incubating total RNA from M. smegmatis with or without MenT1 in the presence of CTP at 37 °C for 1 h; the final RNA and protein concentration was 0.5 µg.µl⁻¹ and 1 µg.µl⁻¹, respectively. Samples were then treated with a demethylase and the library was constructed as described in the methods section. The DNA was subsequently sequenced and the total modified tRNA reads were counted in samples with or without MenT1. E Modification of each specific tRNA form (D) are shown. Results are from two independent experiments.