Dr. Ken Hampel
Our work addresses the fundamental processes by which RNA folds into catalytically active structures. The fact that RNA molecules can function as biological catalysts was first observed twenty years ago. This observation earned its discoverers the Nobel Prize for chemistry in 1989, and has led to a reevaluation of the specific roles served by RNA in essential RNA-protein complexes in nature, such as the ribosome and pre-mRNA splicing complex, the spliceosome. Recent structures of the ribosome make it very clear that peptide bond formation, one of the central biological reactions of life, is catalyzed by RNA.
In order to understand the nature of these fascinating catalysts it is important to appreciate their biophysical and chemical limitations. An important limitation on the correct folding of RNA structures, referred to as the RNA folding problem, is the tendency of any specific RNA primary sequence to fold into a number of different stable conformations. Since structure and function are closely linked, only one of the many possible conformations retains catalytic activity. Inter-conversion of stable conformers is a very slow process, and may thus provide a rationale for the limitation of RNA catalysts (or ribozymes) to in vivo roles where they perform single turnover reactions, or function in obligate RNA-protein complexes. In addition, unlike the diverse repertoire of protein amino acid side-chains, RNA is chemically limited to ribose and the four nucleotide bases, none of which have functional groups suitable for efficient catalysis of acid-base reactions. Nevertheless, naturally occurring ribozymes are capable of accelerating the rate of chemical reactions by many orders of magnitude. How RNA is able to circumvent the problems associated with structural plurality and its limited chemical repertoire are very important questions in modern biochemistry.
We study two small catalytic RNAs, the hairpin and hammerhead ribozymes, as model systems. Both catalyze RNA cleavage reactions through a phosphotransfer reaction pathway. The hairpin ribozyme is composed of two small essential domains that must interact through tertiary contacts to form the catalytic center. We have developed fluorescence and chemical footprinting methods to monitor this process. In addition, we have recently used photocrosslinking analysis to identify a stable tertiary structure that prevents this interaction. Current studies are aimed at determining if this inactive conformation is a part of the native folding pathway for the ribozyme or if it is an example of a the RNA folding problem. Crosslinking can also be used to trap active ribozyme conformations. By this method we have been able to show that the essential base G8 is directed to the active site of the ribozyme during native folding. Functional group changes to G8 interfere with catalysis but not with the essential interdomain interaction. Thus, we believe G8 participates in catalysis directly. A detailed analysis of the functional groups of this base has led to a detailed hypothesis of how G8 functions during the chemistry of hairpin-catalyzed RNA cleavage. Future experiments will be designed to carefully test this and other hypotheses. This integrated approach to analysis of structure and activity has more recently been applied to the hammerhead ribozyme system. We have developed a chemical footprinting system to assay for the formation of a structural intermediate in the catalytic pathway for the hammerhead, and hope to apply this method to distinguish between ribozyme constituents which are required for folding and those that have a role in the chemistry of catalysis.
Walter, N. G., Hampel, K. J., Brown, K. M., and Burke, J. M. Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer. EMBO J. 1998 Apr 15;17(8):2378-2391.
Hampel, K. J., Walter, N. G., and Burke, J. M. The solvent-protected core of the hairpin ribozyme-substrate complex. Biochemistry 1998 Oct 20;37(42):14672-14682.
Hampel, K. J., and Burke, J. M. Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)-EDTA. Methods 2001 Mar;23(3):233-239.