The folding of the hairpin ribozyme: Dependence on the loops and the junction

In its natural context, the hairpin ribozyme is constructed around a four-w ay helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel struc ture to bring them into proximity. In the present study we have compared th e folding of this form of the ribozyme and subspecies lacking either the lo ops or the helical junction using fluorescence resonance energy transfer. T he complete ribozyme as a four-way junction folds into an antiparallel stru cture by the cooperative binding of magnesium ions, requiring 20-40 muM for half-maximal extent of folding ([Mg2+](1/2)) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher ma gnesium ion concentration ([Mg2+](1/2) = 3 mM). Introduction of a G + 1A mu tation into loop A of the ribozyme results in a species with very similar f olding to the simple junction, and complete loss of ribozyme activity. Remo val of the junction from the ribozyme, replacing it either with a strand br eak (serving as a hinge) or a GC(5) bulge, results in greatly impaired fold ing, with [Mg2+](1/2) > 20 mM. The results indicate that the natural form o f the ribozyme undergoes ion-induced folding by the cooperative formation o f an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in th e natural RNA that facilitates the folding of the ribozyme into the active form.