Figure 4. Comparing probes for RNA topoisomerase (RNA Topo) activity.

(a) dPAGE analyses of various structures: monomeric linear (L_h, lane 1), trefoil knot (TK_h, lane 2), and circular (C_h, lane 3) RNA molecules with the sequence for helix-based topological structures; dimeric granny knot (GK_h, lane 4), and circular (C2_h, lane 5) species with the same sequence; and the junction-based negative trefoil knot (TK_j, lane 6). (b) Hypothetical conversions of junction-based (between C_j and TK_j) and helix-based (between C_h and TK_h, and between C2_h, TK2_h and GK_h) topological structures under ‘ideal' RNA Topo condition, when the RNA strand-passage event can freely take place. (c–e) Topological relaxations of TK_j (c), C_h (d) and C2_h (e) catalysed by increasing concentrations of WT E. coli DNA Topo I (30 min incubation at 37 °C). In c,e, the RNA probe substrates (TK_j or C2_h) were 80 nM and Topo I in lanes 1–4 was 40, 80, 160 and 320 nM. In d, the RNA probe substrate (C_h) was 160 nM and Topo I in lanes 1–4 was 80, 160, 320 and 640 nM. In e, C2_h is relaxed to the trefoil knot TK2_h and to GK_h after one and two strand-passage events, respectively. All lanes contain the linear break-down products of the closed RNA structures and they are annotated by LX, in which X represents X-mer.