Structure-Function Studies of Ribozyme and RNA

University Of Colorado, Boulder CO

Investigators

Linked publications & trials

Abstract

This proposal will continue studies on the solution structures of[unreadable] biologically active and potentially biomedically useful RNA enzymes[unreadable] (ribozymes). There has been a major breakthrough in this work during the[unreadable] previous grant period which was the development of methods for[unreadable] isotopically labeling RNAs and subsequent application of the powerful[unreadable] techniques of multi-dimensional heteronuclear magnetic resonance to the[unreadable] solution structure determination of RNAs. The ribozymes that will be[unreadable] studied include: i) a hammerhead ribozyme; ii) a hairpin ribozyme; and[unreadable] iii) a lead-dependent self-cleaving ribozyme termed the leadzyme. These[unreadable] ribozymes can all perform site-specific cleavage of a substrate RNA with[unreadable] multiple turnover. The RNAs will be 13C/15N labeled and then a variety of[unreadable] multi-dimensional heteronuclear magnetic resonance experiments will be[unreadable] used to assign the proton, carbon and nitrogen resonances in these[unreadable] molecules. Once resonance assignments have been made 2D, 3D, or 4D[unreadable] heteronuclear NOESY-type experiments will be performed to obtain proton-[unreadable] proton internuclear distances. Dihedral angle constraints will also be[unreadable] obtained from other multi-dimensional NMR experiments. This distance and[unreadable] dihedral angle information will be used as input for a distance geometry[unreadable] algorithm or constrained molecular dynamics calculations to generate[unreadable] three-dimensional structures consistent with the NMR data. The hammerhead[unreadable] and hairpin ribozymes have site-specific RNA endonuclease activity and[unreadable] have been shown to efficiently cleave specific sites in target RNAs in[unreadable] vitro. There have also been several in vivo applications of these[unreadable] ribozymes as RNA cleavage reagents against specific mRNAs, or as potential[unreadable] antiviral agents that target the genome of an RNA virus such as HIV-l. The[unreadable] structural information obtained from the NMR experiments proposed here[unreadable] will provide valuable data for the design of improved ribozymes and will[unreadable] be extremely helpful in interpretation of the wealth of biochemical,[unreadable] kinetic and mutagenesis data on these ribozyme systems.[unreadable]