Fig. 5: Phage-activated RorSlfn5 cleaves the tRNA anticodon arm.
a,b, Position-specific mapping of internal 3′ ends (a) and 5′ ends (b) in tRNALys(UUU) in T5 phage-infected cells expressing RorSlfn5. The data are shown as the mean of three biological replicates. c, Alignment of sequencing reads to the lysW gene of MG1655 cells. One representative replicate from three biological replicates is shown. d, SEC of affinity-purified complex of RorSlfn5 and T5.142 (left). The elution fraction highlighted with a light blue rectangle was analysed with SDS–PAGE (right). The full SEC profile can be found in Extended Data Fig. 7. e, tRNA cleavage assays with 100 nM tRNALys(UUU) and 100 nM RorSlfn5 in the presence of T5.142 trigger (2 µM) and Mn2+ (2 mM). f, The same as in e but with 2 mM Mg2+, 2 mM Mn2+ or no metal and 10 mM EDTA. All shown assays were reproduced independently three times with similar results. g, Potential RorSlfn5 cut sites (triangles) in the anticodon arm of tRNALys(UUU), according to RNA-seq data in a and b. The red triangle shows the cut site supported by cleavage assays in e and f. h, A proposed model for RorSlfn5-mediated tRNA cleavage in vivo and subsequent host exoribonuclease (exoRNase) trimming of the anticodon loop.
