RNA molecules with structure-dependent functions play important roles throughout molecular biology. The broad, long-term objective of the proposed research is to better understand the structural and catalytic properties of RNA. The focus of the first two specific aims is on a ribozyme that polymerizes RNA using the same reaction as that of protein enzymes that replicate RNA. This reaction is among the most complex reactions known to be catalyzed by RNA. At the heart of the polymerase is a ligase ribozyme, which is amenable to high-resolution structural studies. When outside the context of the rest of the polymerase, the ligase promotes a reaction related to but simpler than that of the polymerase, and notable as being among the fastest known to be catalyzed by RNA. The structure and activity of the polymerase will be examined and enhanced with a set of experiments that involve biochemical, crystallographic, engineering, and combinatorial approaches, with the specific aims of i) characterizing the structure of the polymerase ribozyme (including the atomic-resolution structure of the ligase, which constitutes the polymerase catalytic care; and ii) improving the efficiency of RNA-catalyzed RNA polymerization. The third specific aim is to search for intersection sequences that link aminoacylase and kinase ribozymes.
This aim addresses the idea that the extreme conformational dexterity of RNA might enable new ribozyme folds to emerge from pre-existing folds by means of evolutionary paths that do not include inactive, non-functional sequences. These experiments will contribute to our fundamental knowledge of RNA molecular biology. They will describe and extend known limits of RNA catalytic function, begin to explain how RNA can achieve such rapid and complex function, provide insights into how new RNA folds can emerge during evolution, and speak to the ability of RNA to catalyze self-replication-the central presumption of current theories of the early evolution of life. The high-resolution structure of the ligase ribozyme will also be useful for others who are attempting to develop highly sensitive clinical diagnostics based on this ribozyme. More generally, innovations and insights from these experiments will enable and inspire those seeking to isolate new ribozymes with efficient and complex functions, including ribozymes useful as diagnostics, therapeutics, and research tools.
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