LOOP-SIZE VARIATION TO PROBE A BENT STRUCTURE OF A HAIRPIN RIBOZYME

A two-stranded hairpin ribozyme, derived from the catalytic center of the negative strand of the tobacco ring spot virus satellite RNA, cata lyzes the cleavage and joining of RNA fragments. In order to probe the bent structure in hairpin ribozymes, complexes were constructed by jo ining a substrate to the shorter strand of this ribozyme, using multip le units of 1,3-propanediol phosphate at the junction. Active conforma tions of the complex, in the presence of magnesium ions for substrate cleavage, were estimated by increasing the number of non-nucleotidic l inkers. Significant cleavage was observed in the molecule with three l inker units and was relatively increased in molecules bearing 4 and 5 linkers. Complexes with 7, 10, and 13 linkers showed cleavage rates of 3.6-, 7.3-, and 8.7-fold of that for the complex with 5 linkers. The effects of the magnesium chloride concentration were greater in molecu les with 5 and 7 linkers as compared to those with 10 and 13. Extra sp ace in the junction was shown to be required to form an active structu re of the ribozyme-substrate complex. A bent structure in the hairpin ribozymes caused by the extra space has been proposed. A model of the complex with a bend was constructed by joining the junctions with link ers.