Catalytically active geometry in the reversible circularization of ‘mini-monomer’ RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA

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Abstract
The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(−)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the llgatlon of substrate cleavage products and that ollgoribonucleotldes, termed ‘mini-monomers’ and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reverslbly. The kinetics of llgation and cleavage of one such mini-monomer was consistent with a simple unlmolecular reaction at some temperatures. Evidence suggests that the circular llgatlon product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unlmolecular reaction but did when incubated with second, trans-acting ollgoribonucleotldes that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular llgation. We suggest the term ‘paperclip’ to represent the arrangement of RNA strands In the region of sTobRV(−)RNA that participates in the cleavage and ligation reactions.