EXCEED THE SPACE PROVIDED. Understanding mechanisms of RNA catalysis remains an intriguing challenge, one that has grown in significance since the recent demonstration that the ribosome is a ribozyme. Several ribozymes catalyze the same reversible RNA cleavage reaction but adopt different structures and exploit distinct kinetic and catalytic mechanisms. Comparison of these mechanisms provides insight into the diversity of catalytic strategies that are available to RNA enzymes. The unique pH and metal ion dependencies of hairpin ribozyme catalysis was the first sign that ribozymes can accomplish catalysis exclusively through the use of RNA functional groups with no requirement for direct coordination of metal cations to phosphate, ribose or water oxygens. Until now, the difficulty of distinguishing structural from catalytic effects of experimental manipulations seriously limited studies of the hairpin catalytic mechanism. Two recent developments have transformed the field in this regard. First, a crystal structure has been solved for a hairpin ribozyme complex with a substrate analog that provides the first high resolution view of the functional groups that comprise the active site. Second, hairpin ribozyme variants have been developed that allow catalytically important nucleotides to be modified without disrupting the functional structure. The proposed research will develop a detailed model for the hairpin ribozyme catalytic mechanism by elucidating the roles of specific RNA functional groups in catalytic chemistry and active site architecture. We will test whether ribozyme functional groups mediate general acid base catalysis by examining pH-rate profiles for reactions of ribozyme variants missing specific active site nucleobases or with active site nucleobases replaced by nucleobase analogs that differ in acid or base strength. Preliminary data suggest that cationic nucleobases might provide electrostatic stabilization to negative charge developing in the transition state or stabilize oxyanion nucleophiles or leaving groups. We will examine this notion using an abasic ribozyme rescue strategy and through covalent incorporation of nucleobase analogs that differ in the propensity to undergo ionization. Finally, we will examine how structural and biochemical features of the active site influence catalysis through positioning and orientation of reactive groups. PERFORMANCE SITE ========================================Section End===========================================

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Jones, Warren
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Scripps Research Institute
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Viladoms, Julia; Fedor, Martha J (2012) The glmS ribozyme cofactor is a general acid-base catalyst. J Am Chem Soc 134:19043-9
Viladoms, Julia; Scott, Lincoln G; Fedor, Martha J (2011) An active-site guanine participates in glmS ribozyme catalysis in its protonated state. J Am Chem Soc 133:18388-96
Cottrell, Joseph W; Scott, Lincoln G; Fedor, Martha J (2011) The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine. J Biol Chem 286:17658-64
Watson, Peter Y; Fedor, Martha J (2009) Determination of intracellular RNA folding rates using self-cleaving RNAs. Methods Enzymol 468:259-86
Liu, Lu; Cottrell, Joseph W; Scott, Lincoln G et al. (2009) Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme. Nat Chem Biol 5:351-7
Fedor, Martha J (2008) Alternative splicing minireview series: combinatorial control facilitates splicing regulation of gene expression and enhances genome diversity. J Biol Chem 283:1209-10
Cottrell, Joseph W; Kuzmin, Yaroslav I; Fedor, Martha J (2007) Functional analysis of hairpin ribozyme active site architecture. J Biol Chem 282:13498-507
Da Costa, Carla P; Fedor, Martha J; Scott, Lincoln G (2007) 8-Azaguanine reporter of purine ionization states in structured RNAs. J Am Chem Soc 129:3426-32
Kuzmin, Yaroslav I; Da Costa, Carla P; Fedor, Martha J (2004) Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue. J Mol Biol 340:233-51
Lebruska, Lori L; Kuzmine, Iaroslav I; Fedor, Martha J (2002) Rescue of an abasic hairpin ribozyme by cationic nucleobases: evidence for a novel mechanism of RNA catalysis. Chem Biol 9:465-73

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