Extended Data Fig. 5: Biochemical characterization of ωRNA.
From: Cryo-EM structure of the transposon-associated TnpB enzyme
(a) Urea-PAGE analysis of the purified TnpB–ωRNA complex. Although we co-expressed TnpB with a 247-nt ωRNA, the purified TnpB was bound to heterogenous 100–160-nt parts of the ωRNA. (b) The sequence of full-length ωRNA (−231G to 16C). The probe positions used in the northern blotting analysis are shown in orange. Two GAAC sites that could generate a pGAACp fragment by RNase A digestion are indicated in red. (c) Northern blotting of ωRNA. Total RNAs prepared from E. coli wild-type cells (lane 1), ωRNA expressing cells (lane 2), ωRNA and MBP co-expressing cells (lane 3), ωRNA and TnpB co-expressing cells (lane 4), the in vitro transcript of ωRNA (lane 5), and the ωRNA extracted from TnpB (lane 6) were resolved by 10% denaturing PAGE and stained with GelGreen (left panel) or subjected to northern blotting (right panels, Probes I–V). 6S RNA (180-nt), 5S rRNA (120-nt), tRNAs (76 to 93-nt) in total RNA and 50-nt, 100-nt, and 300-nt RNA markers are indicated (lane M). (d) Collision-induced dissociation spectrum of the pGAACp fragment from ωRNA digested by RNase A. The divalent negatively charged ion of pGAACp was used as the precursor ion for CID. The product ions in the CID spectrum are assigned on the sequence.
