Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

^*Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT 05405; and ^‡Nucleic Acids Products Supply (NAPS), Göttingen GmbH, Rudolf-Wissell Str. 28, 37079 Göttingen, Germany

Edited by George Bruening, University of California, Davis, CA, and approved March 23, 1998 (received for review December 3, 1997)

Abstract

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous 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 inactive form of the complex was trapped by the addition of T4 RNA ligase to 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 quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

Footnotes

↵ † Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
↵ § To whom reprint requests should be addressed. e-mail: jburke{at}zoo.uvm.edu.
This paper was submitted directly (Track II) to the Proceedings Office.
↵ ¶ Under the reaction conditions described in Materials and Methods, ligation of the ribozyme-substrate complex catalyzed by T4 RNA ligase followed multiphasic kinetics, probably because of its complicated reaction mechanism (23). The weighted average rate for this reaction (sum of the exponential rate of each phase multiplied by the corresponding amplitude) was 0.03–0.04 min⁻¹. This ligation rate is significantly faster than the dissociation rate measured for inactive ribozyme-substrate complexes (0.01 min⁻¹; see ref. 13). Therefore, a quantitative ligation of the inactive conformation can be expected. T4 DNA ligase is also able to efficiently ligate RNA strands in duplex structures, but this reaction is strongly dependent on the presence of a complementary DNA strand acting as a bridge (24).