The biological function of RNA is intimately related to its three-dimensional structure. A tertiary RNA structure forms through a network of interactive modules. Independently stable tertiary structures can be linked together in a RNA enzyme to improve substrate binding and catalysis. The long-term objective of this proposal is to design stable tertiary RNA structures that can be incorporated as an essential part of an enzyme. The ribozyme consists of two domains that are independently stable. The first aim is to study how one of the two domains is stabilized by small, modular structures. This domain is proposed to contain two tertiary structural modules, one involved in function and the other involved in stability. Two naturally occurring modules of different sequences will be exchanged to demonstrate this two- module concept. The quantitative effect of altering the relative orientation and flexibility of the functional and stability module will be determined. A collection of the stability modules will be generated by in vitro section and the relative thermodynamic contribution of the selected modules will be determined in anticipation of the rational design. The second aim is to study how this ribozyme uses its two-domain composition to carry out catalysis. A new model of catalysis by this ribozyme will be tested in which both domains undergo a conformation change to increase the fraction of an obligatory ribozyme-substrate complex along the reaction pathway. The junction region of these domains will be modified to correlate the formation of this obligatory ribozyme-substrate complex to the conformation adjustment of these domains. Spectroscopic detection of the changes in the domain-domain orientation will be attempted by fluorescence resonance energy transfer. The third aim is to generate "custom-made" ribozymes that recognize and cleave RNA structures or even RNA-protein complexes of choice. This goal will be accomplished by a combination of rational design and in vitro section and is made possible through our extensive knowledge on binding and catalysis of this ribozyme. These results will reveal how to stabilize a complex RNA structure, how to employ multiple domains to improve RNA catalysis and how to isolate ribozymes that can cleave any RNA structure or an RNA-protein complex.

R01GM052993

1995-08-04

2004-07-31