Research

Structure of ribonucleic acids (RNAs) and proteins involved in catalysis and gene regulation

My laboratory is engaged in a broad research program involving structural biology, molecular biology, and drug-discovery. Using high-resolution structural biology we wish to establish the molecular basis of recognition used by ribonucleic acids (RNAs) and proteins. Currently proteins are the focus of most drug discovery efforts. However RNA and RNA-protein (RNP) interactions play several central roles in many biological and diseases processes from catalysis to gene regulation. With better understanding of RNA structure and an improvement in biophysical and biochemical techniques available to study RNA, new research seeks to exploit RNA and RNP particles as novel targets for pharmaceutical development. The current projects in my laboratory are focused on establishing the technologies needed for structured-assisted drug-discovery using the derived principles of molecular recognition. The major disease targets are cancer and heart. Our workhorse in the past three years has been the group II intron ribozyme project funded by the National Science Foundation and in collaboration with Dr. Rick Padgett (Molecular Genetics). The techniques developed in the execution of this project are now being applied to probing the structural basis of gene specific translational silencing and endoribonuclease activity.

Structure and Mechanism of Group II Intron RNA Enzyme

Catalytic enzymatic function is essential to life and RNA enzymes such as group II introns splice the RNA within which they reside without the help of other proteins. They also cleave and insert themselves into double-stranded DNA using the ribozyme active site and protein co-factors. Splicing is an essential biologic process for the expression of most eukaryotic genes by the removal of intron RNA from coding sequences because accurate splicing is needed to avoid mutations that lead to translation of aberrant mRNAs and create protein diversity in metazoan organisms. This pre-mRNA splicing in eukaryotes is catalyzed by a large macromolecular ribonucleoprotein machine called the spliceosome that uses a chemical reaction pathway similar to those used by self-splicing group II introns. Understanding the structural basis for the molecular mechanism of splicing is therefore of great physiological and pathological importance. We have adapted a tripartite model system comprising domain 5 (D5), domains 1, 2, and 3 (D123), and substrates from the brown algae Pylaiella littoralis (PL) that allows us to use D5 as an “enzymatic reagent” to elucidate how conserved nucleotides in D5 are positioned within the active site to effect binding and catalysis. We have almost completed with our initial goal of obtaining the structure of D5 by itself. Next we have begun developing a FRET-based enzymatic assay to probe the interaction of D5 with substrates. Finally we hope to make use of the excellent array of ultra-high field NMR instruments (600-900 MHz) to resolve the 160 kDa complex of D5 bound to D123 and a substrate. Ultimately, our structural studies will serve as a model for splicing reaction of the large spliceosomal machinery.