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. 2021 Jul 29;17(7):e1009675. doi: 10.1371/journal.pgen.1009675

Fig 1. Exonuclease T digests the nucleotide A from 3´-end of uncharged tRNA.

Fig 1

(A) Illustration of the enzymatic method to differentiate charged and uncharged tRNAs. (B) Uncharged tRNALeu digested by exonuclease T is 1 nt shorter than charged tRNALeu in a northern blot. Total RNA from HeLa cells was treated with or without N-acetylation, with or without exonuclease T digestion, and loaded on a 15% urea acrylamide gel. Radiolabeled RNA marker with various sizes were loaded in the left lane. A northern blot was performed using the tRNALeu probe. (C) N-acetylation protects Leu-tRNALeu from deacylation and digestion with exonuclease T treatment. E. coli acetylated-Leu-tRNALeu (inverted triangle). Leu-tRNALeu (solid circle), uncharged tRNALeu (open circle) were prepared and subjected to exonuclease T digestion at room temperature. Samples were taken and quenched on ice at time points of 0, 5, 10, 30, 60 mins. Northern blots were used to detect the 1nt size difference after digestion. Levels of digested versus undigested tRNAs were quantified by Quantity One with three independent replicates.