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Scientific Report 2008

Chemical Physiology

Mechanisms of RNA Assembly and Catalysis

M.J. Fedor, J.W. Cottrell, L. Liu, L. Li, P. Watson, S. Zimmerman

Our goal is to understand how RNA enzymes catalyze biological transformations so we can gain basic insights into fundamental aspects of gene expression and build a framework for technical and therapeutic applications in which RNAs are used as targets and reagents. The hairpin ribozyme catalyzes a reversible phosphodiester cleavage reaction that involves in-line attack of the 2′oxygen nucleophile on the adjacent phosphorus to create a trigonal bipyramidal transition state and generates products with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. Structural studies have revealed a network of stacking and hydrogen-bonding interactions that align the reactive phosphate in the appropriate orientation for an SN2-type nucleophilic attack and orient nucleotide base functional groups near the reactive phosphate to facilitate catalytic chemistry (Fig. 1).

Fig. 1. Structure of the hairpin ribozyme active site in a ribozyme complex with a vanadate (green) mimic of the transition state that shows interactions with the catalytically essential G8 and A38 nucleotide bases.

Ribonuclease A is a protein enzyme that catalyzes the same chemical reaction as the hairpin ribozyme but has an active site composed of amino acids rather than nucleotides. Ribonuclease A provides a textbook example of concerted general acid-base catalysis in which a histidine acts as a general base to activate nucleophilic attack by removing a proton from the 2′ hydroxyl while a second histidine protonates the 5′ oxygen-leaving group to facilitate breaking the 5′ oxygen-phosphorus bond. The positions of G8 and A38 nucleobases in the active site of the hairpin ribozyme resemble the orientation of the 2 histidine side chains in the active site of ribonuclease A, leading to the suggestion that G8 and A38 might also mediate general acid-base catalysis. With an acid ionization constant near 6.5, significant fractions of histidine residues are in both protonated and deprotonated states, so histidines are adept at accepting and donating protons at neutral pH. However, adenosine and guanosine undergo ionization only at pH extremes, at least as free nucleosides in solution, a characteristic that seems to make them poorly suited for mediating proton-transfer reactions at neutral pH.

We are using fluorescent nucleotide analogs to learn whether some feature of the ribozyme active site alters the ionization equilibria of adenosine or guanosine relative to the ionization behavior of the nucleotide bases in solution and enhances their ability to serve as general acid-base catalysts. An 8-azapurine analog of guanine has a high fluorescent quantum yield when the N1 position is deprotonated and a low fluorescent quantum yield when N1 is protonated (Fig. 2). A hairpin ribozyme in which G8 has been replaced by 8-azaguanine has full catalytic activity, evidence that an 8-azapurine analog can indicate the ionization equilibria of G8 without perturbing the ribozyme active site. The protonation-dependence of the intensity of fluorescence emission enabled us to calculate ionization equilibria from changes in emission intensity with pH.

Fig. 2. 8-Azaguanine, an analog of guanine, has a high fluorescent quantum yield when the N1 position is deprotonated at high pH and low fluorescence intensity when N1 is protonated at neutral pH. Ribozymes with 8-azaguanine substituted for G8 enable us to measure any change in ionization state that might occur within the context of a functional active site.

We found that fluorescence is quenched 10- to 100-fold when 8-azaguanine is incorporated into base-paired RNA, as reported previously for other fluorescent nucleobase analogs. The apparent ionization equilibrium for 8-azaguanine that we determined from the pH dependence of fluorescence intensity in the context of a perfectly paired duplex RNA is shifted in the basic direction, consistent with the idea that removing a proton and introducing a negative charge is more difficult in a stacked RNA helix than in a free nucleotide in solution. Strikingly, the ionization equilibria of 8-azaguanine in the context of the hairpin ribozyme active site also is shifted in the basic direction relative to the ionization equilibria of 8-azaguanosine in solution. Thus, our results provide no support for the idea that G8 is more adept at general acid-base catalysis in a ribozyme active site than it would be as a free nucleoside in solution and suggest alternative roles for G8 in positioning and electrostatic stabilization.


Martha J. Fedor, Ph.D.
Associate Professor

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