Figure 5.

Lasso ALR562-2 binding to linear TNF685 and a circular target (CMT). (a) Products of self-processing of internally ³²P-labeled ALR562-2 in transcription buffer containing 6 mM Mg²⁺ (lane 1) and in buffer containing 50 mM Tris–HCl, 10 mM MgCl₂, 20% formamide (lane 2). Lane 3: formation of the strong complex between ALR562-2 (from lane 2) and nonradioactive TNF685 target provided in excess. Lane 4: products of dissociation of the complex from Lane 3 upon addition of 50% formamide and 10 mM EDTA and post-incubation for 2 min at 90°C. The products were analyzed by 6% d-PAGE and bands were assigned to Lasso-target complexes as well as unbound circular (CL), unprocessed (UPL), half-processed (HPL) and linear (LL) Lasso forms as indicated. (b) Demonstration of topological linkage between CMT and Lasso ALR562-2. Internally ³²P-labeled Lasso ALR562-2 was incubated in TM–formamide buffer containing 50 mM Tris–HCl (pH 7.5), 10 mM MgCl₂, 20% formamide (lanes 1–3) or 50 mM Tris–HCl (pH 7.5), 10 mM EDTA, 20% formamide (lanes 4–6) for 2 h at 37°C either alone (lanes 1 and 4) or with the CMT target (lanes 2–3 and 5–6). Samples in lanes 3 and 6 were additionally postincubated at 95°C for 5 min followed by quenching on ice. The products were analyzed by denaturing 6% PAGE and assigned to complexes of CMT with various forms of the Lasso (HPL-CMT, LL-CMT and CL-CMT) as well as unbound circular (CL), unprocessed (UPL), half-processed (5′-HPL and 3′-HPL) and linear (LL) forms as indicated.