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. 2020 Jul 29;6(31):eabb6651. doi: 10.1126/sciadv.abb6651

Fig. 4. Toxin MenT3 adds pyrimidines to the 3′-CCA acceptor stem of tRNA.

Fig. 4

(A) Radiolabeled E. coli tRNATrp was incubated with 1, 0.1, 0.01, or 0.001 μg of MenT3 WT or no toxin (−) for 20 min at 37°C in the presence of unlabeled GTP, ATP, UTP, or CTP. Extended products are indicated with arrowheads throughout all panels. (B) Radiolabeled E. coli tRNATrp was incubated with 1, 0.1, or 0.01 μg of MenT3 WT or MenT3(D80A) with CTP or UTP, as per conditions in (A). (C) Incubation of radiolabeled E. coli tRNATrp with 1, 0.1, 0.01, or 0.001 μg of MenT3 WT or MenT3(K189A), with CTP, UTP, or a mixture of both, as per conditions in (A). (D) Radiolabeled E. coli tRNATrp preparations, made with or without a 3′-CCA motif, were incubated with 1, 0.1, or 0.01 μg of either MenT3 WT, MenT3(K189A), or no toxin (−), for 20 min at 37°C in the presence of unlabeled UTP or CTP. Note that the (−) CCA lanes have been overexposed to equalize intensity to the (+) CCA lanes of the same gel. Assays of the individual WT and MenT3 substitution proteins and tRNATrp ± CCA substrates shown in (A) to (D) were performed between two and four times.