A synthetic strand of RNA has been designed so that it can adopt two different topological states (a circle and a trefoil knot) when ligated into a cyclic molecule. The RNA knot and circle have been characterized by their behavior in gel electrophoresis and sedimentation experiments. This system allows one to assay for the existence of an RNA topoisomerase, because the two RNA molecules can be interconverted only by a strand passage event. We find that the interconversion of these two species can be catalyzed by Escherichia coli DNA topoisomerase III, indicating that this enzyme can act as an RNA topoisomerase. The conversion of circles to knots is accompanied by a small amount of RNA catenane generation. These findings suggest that strand passage must be considered a potential component of the folding and modification of RNA structures.The role of type I topoisomerases (1) is key in the cellular metabolism of DNA. These enzymes are involved intimately in replication, in transcription, and in the maintenance of torsional stress in the genome. The importance of RNA within the cell is well-recognized, as mRNA, as tRNA, in ribosomes, and in processing roles. RNA molecules that have been characterized by x-ray crystallography to high resolution contain hairpins and pseudoknots (2-4). The linear single-stranded character of cellular RNA has generally led to the assumption that functional RNA structures can be achieved without the requirement for strand passage activities to solve problems in RNA molecular topology. Hence, in contrast to DNA, the cellular need for an RNA topoisomerase has not seemed compelling. Nevertheless, it has been shown recently that Escherichia coli DNA topoisomerase III (topo III) (5, 6) is capable of cleaving RNA molecules to produce a protein-RNA adduct (7). Here, we demonstrate that topo III is capable of catalyzing strand passage operations that interconvert synthetic RNA circles and knots.Previously, we have constructed DNA knots by cyclizing synthetic single-stranded molecules using a pairing motif of the form X-S-Y-S-X'-S-Y'-S-, where X and Y represent 11 or 12 nucleotides, respectively, X' and Y' are their Watson-Crick complements, and S is a single-stranded spacer region consisting of dTn, where n ranges from 6 to 15 (8-12). Knots formed from this motif are typically trefoil knots with negative nodes. However, by using sequences in the X or Y domains capable of forming left-handed Z-DNA (13), we have also been able to form figure eight knots (9, 10) that are topological rubber gloves (11), as well as trefoil knots with positive nodes (12). We have demonstrated recently that these 104 nt DNA knots and their corresponding circle can be interconverted by E. coli DNA topoisomerase I (topo I) and by topo III (14). We have used the same motif to construct a 104-nt RNA trefoil knot, with negative nodes. A 40-nt DNA linker (incompatible with knot formation) is annealed to the molecule, and it is ligated together to form an RNA circle, which survives treatment with DNase. In the other ...