Fig. 3.

RloC-expressing cells manifest ACNase activity.

A. Total RNA samples isolated from cells expressing PrrC (odd lanes) or RloC (even lanes) were separated by denaturing polyacrylamide gel electrophoresis as such (lanes 1, 2) or after being 5′-end-labelled by T4 Pnk (lanes 5, 6). The non-labelled RNA fractions were also further incubated with T4 Pnk and Rnl 1 (lanes 3, 4) and then ligated with Rnl1 (lanes 7, 8). The gel was then stained with ethidium bromide (lanes 1–4) or autoradiographed (lanes 5–8). 33mers are 5′-cleavage products generated by either ACNase. 43mers are 3′-cleavage products generated by PrrC, ∼42mers and ∼52mers 3′-cleavage products generated by RloC. Band a contains the ligated PrrC cleavage products, bands a* and b contain the RloC counterparts, and bands c represents presumable internally ligated (circular) cleavage products of either ACNase.

B. Scheme describing the cleavage of the ACNase substrate, 5′-end-labelling of the 3′-cleavage product, the subsequent ligation and the release of the labelled nucleotides from the labelled fragments or ligated-back molecules by the indicated nucleases.

C. 2D TLC of radiolabelled nucleotides released by nuclease P1 (panels I–VII) or RNase T2 (panels IX–XI) from the indicated labelled RNA preparations. The 5′-end-labelled 43mers, ∼42mers and ∼52mers (Fig. 4A, lanes 1, 2), their ligated-back derivatives of bands a, a* and b (lanes 3, 4) and the circularized forms of RloC's products (lane 3) were digested by nuclease P1 and the released radiolabelled nucleotides separated by 2D TLC, as indicated. 5′-NMP markers are shown in panel VIII. The 3′-NMPs released from the indicated ligated-back derivatives by RNase T2 were similarly separated. The identity of U⁸ (panels I, II) was ascertained by subsequent separation on PEI-cellulose TLC (not shown). X¹–X⁵ indicate apparent modified or hypomodified nucleotides that were not identified.

D. Identification of tRNA species cleaved by PrrC or RloC. The 5′-end-labelled 43mers generated by PrrC (Fig. 4A, lane 1) or RloC's ∼42mers (Fig. 4A, lane 2) were hybridized to dot blots containing antisense DNA oligonucleotides corresponding to the indicated E. coli tRNA species described by Jiang et al. (2001) and in Table S2.