STRUCTURAL BASIS FOR HETEROGENEOUS KINETICS - REENGINEERING THE HAIRPIN RIBOZYME

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biph asic kinetics, and chase experiments show that the slow phase of the r eaction results from reversible substrate binding to an inactive confo rmational isomer. To investigate the structural basis for the heteroge neous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inact ive form of the complex was trapped by the addition of T4 RNA ligase t o a cleavage reaction, resulting in covalent linkage of the 5' end of the substrate to the 3' end of the ribozyme and in selective and quant itative ablation of the slow kinetic phase of the reaction. This resul t indicates that the inactive form of the ribozyme-substrate complex c an adopt a conformation in which helices 2 and 3 are coaxially stacked , whereas the active form does not have access to this conformation, b ecause of a sharp bend at the helical junction that presumably is stab ilized by inter-domain tertiary contacts required for catalytic activi ty. These results were used to improve the activity of the hairpin rib ozyme by designing new interfaces between the two domains, one contain ing a non-nucleotidic orthobenzene linkage and the other replacing the two way junction with a three-way junction. Each of these modified ri bozymes preferentially adopts the active conformation and displays imp roved catalytic efficiency.