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Molecular Biology
Mechanisms of RNA Assembly and Catalysis
M.J. Fedor, E.M. Calderon,
J.W. Cottrell, C.P. Da Costa, J.W. Harger, Y.I. Kuzmin, E.M. Mahen, R.S. Yadava
The
ways that RNA enzymes accomplish catalysis are of considerable interest, particularly because
of compelling evidence that protein synthesis itself is an RNA-catalyzed reaction. Like proteins,
RNAs assemble into ordered structures that can facilitate catalysis by positioning reactive
groups in an optimal orientation. However, ribonucleosides lack the chemical versatility of
amino acids. At first, it seemed that all RNA enzymes compensated for this lack of chemical versatility
by recruiting metal cation cofactors. Then researchers found that a catalytic RNA, the hairpin
ribozyme, catalyzes a reversible phosphodiester cleavage reaction in the absence of metal cations
(Fig. 1). The ability of the hairpin ribozyme to accomplish catalysis without metal cations raised
the possibility that RNA functional groups might participate directly in catalytic chemistry.
Our goals are to determine which parts of the hairpin ribozyme contribute to catalytic activity
and to understand the chemical basis of this activity.
 |
| Fig. 1. Chemical mechanism of RNA cleavage mediated by the family
of small catalytic RNAs that includes the hairpin ribozyme. Cleavage proceeds through an SN2-type
mechanism that involves in-line attack of the 2¢
oxygen nucleophile on the adjacent phosphorus. Breaking of the 5¢
oxygen-phosphorus bond generates products with 5¢
hydroxyl and 2¢,3¢-cyclic
phosphate termini. |
Biochemical and structural studies implicate
2 active-site nucleobases, G8 and A38, in the catalytic mechanism. High-resolution structures
of the active site led to a general acid-base model in which G8 removes a proton to activate the attacking
2¢ hydroxyl nucleophile,
and A38 protonates the 5¢ oxygen-leaving
group. We developed an approach involving exogenous nucleobase rescue of abasic ribozymes to
probe potential catalytic roles of G8 and A38. In this approach, abasic phosphodiester linkages
replace individual active-site nucleobases. Characterization of the residual activity of abasic
variants provides valuable clues about the function of the missing nucleobase.
We
found that activity increases with increasing pH in reactions with unmodified ribozymes, consistent
with the idea that the ionization state of a general base catalyst could be important for activity.
Abasic ribozymes lacking G8 are much less active than are unmodified ribozymes, as expected if
G8 participates in catalysis. However, the residual activity of a variant lacking G8 has the same
pH dependence as reactions of unmodified ribozymes. This similarity in pH dependence for unmodified
and abasic ribozyme reactions argues against a role for G8 as a general base catalyst because deprotonation
of G8 could not account for the pH transition in the abasic variant. On the other hand, loss of A38
shifts this pH transition by several pH units, evidence that a deprotonation event associated
with A38 could be important for activity.
The activity that is lost when G8 is replaced
by an abasic residue can be rescued by providing certain nucleobases in solution. Rescue of activity
by exogenous nucleobases increases sharply with decreasing pH, indicating that a step that requires
protonation becomes rate determining for rescue. Several models of the rescue mechanism could
explain this pH dependence. In a general acid catalysis model, the rescuing nucleobase protonates
the 5¢ oxygen-leaving group.
In an electrostatic stabilization model, interactions with the rescuing nucleobase counter
negative charge that develops in the transition state. Alternatively, protonation might simply
facilitate bindings of exogenous nucleobases to restore active-site geometry.
Detailed analyses of exogenous nucleobase
rescue allow us to begin to distinguish among these models. Rescue increased with decreasing pH
for both cleavage and the reverse reaction of ligation, arguing against a role for the rescuing
nucleobase as a general acid because a nucleobase that contributes general acid catalysis in the
cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration
dependence of nucleobase rescue at high and low pH indicated that protonation promotes catalysis
within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the
ground state. Taken together, these results support a novel electrostatic stabilization mechanism
in which exogenous nucleobase binding counters negative charge that develops in the transition
state (Fig. 2).
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| Fig. 2. Model of electrostatic stabilization by a cationic nucleobase.
The exogenous nucleobase stacks between A7 and A9 in the pocket left by the G8 deletion, restoring
active-site architecture. The amidine group of the rescuing nucleobase forms hydrogen bonds
with 2¢ hydroxyl and phosphoryl
oxygens. These interactions stabilize negative charge that develops in the transition state
and position reactive groups in the orientation appropriate for an SN2 in-line nucleophilic
attack. |
Publications
Fedor, M.J.
Determination of kinetic parameters for hammerhead and hairpin ribozymes. Methods Mol. Biol.
252:19, 2004.
Kuzmin, Y.I., Da Costa, C.P., Fedor,
M.J. Role of an active site guanine in hairpin ribozyme catalysis
probed by exogenous nucleobase rescue. J. Mol. Biol. 340:233, 2004.
Yadava, R.S., Mahen, E.M., Fedor,
M.J. Kinetic analysis of ribozyme-substrate complex formation
in yeast. RNA 10:863, 2004.
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