News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
Intracellular RNA Assembly and Catalysis
M.J. Fedor, C.P. Donahue, H.A. Erlacher, S. Nesbitt
The mechanisms used by some RNA enzymes to catalyze biochemical reactions
in vitro are now as well understood as the mechanisms used by their protein counterparts.
Despite the importance of RNA-mediated reactions in RNA processing and translation,
however, it remains unclear how reaction pathways defined for RNA enzymes in
vitro relate to RNA-mediated reactions in living cells. To address this gap,
we have undertaken a quantitative analysis of a simple RNA-catalyzed reaction
The hairpin ribozyme is composed of 2 helix-loop-helix segments with the
reactive phosphodiester located in one of the loops (Fig. 1).
This ribozyme cleaves phosphoester bonds in a reversible reaction that generates
5´ hydroxyl and 2´,3´-cyclic phosphate termini. In contrast to
the requirement for high concentrations of metal cations typical of other ribozymes,
hairpin catalysis requires only buffer and counterions. Because of its unique
catalytic strategy, the hairpin ribozyme is particularly well suited to structure-function
studies in vivo where concentrations of metal cations are low.
We expressed hairpin ribozymes in yeast as chimeric self-cleaving mRNAs.
Chimeric mRNAs containing self-cleaving hairpin ribozymes were compared with
the same mRNAs containing an inactivating mutation in the hairpin sequence. Chimeric
mRNAs containing mutant hairpin ribozymes decay through endogenous mRNA degradation
pathways. Self-cleaving mRNAs degrade through the normal pathway but also disappear
through self-cleavage. Consequently, the difference in decay rates between mutant
and self-cleaving RNAs allows us to calculate the intracellular cleavage rate
In vivo, ribozymes self-cleave at rates similar to the rates of self-cleavage
measured in vitro. A ribozyme mutation that slows cleavage in vitro also slows
intracellular cleavage by a similar amount. Thus, the reaction pathway and the
rate-determining steps for hairpin ribozyme--mediated cleavage are fundamentally
the same in vitro and in vivo.
Cleavage kinetics monitor assembly and dissociation steps as well as catalytic
steps in the reaction pathway, so this system provides a unique opportunity to
assess RNA structure and dynamics inside living cells. To assess the requirements
for RNA helix assembly in vivo, we compared intracellular self-cleavage rates
for a series of hairpin ribozymes with helix I sequences as small as 1 bp. Helices
shorter than 3 bp are too weak to support full activity in vitro. We found that
the same situation is true in vivo, arguing against any significant stabilization
of short RNA helices in vivo. Under standard conditions in vitro, cleavage is
reversed rapidly by ligation when cleavage products remain bound to the ribozyme.
Consequently, ribozymes with products bound in helices with more than 6 bp have
sharply reduced self-cleavage rates in vitro because slow release of product
becomes rate determining. In contrast, ribozymes with products bound through
as many as 18 bp self-cleave at nearly normal rates in vivo. The failure of large
products to inhibit self-cleavage could be explained if intracellular factors
promote rapid helix dissociation. Alternatively, the balance between cleavage
and ligation might not favor ligation in vivo as it does in vitro. The propensity
of the hairpin ribozyme to catalyze ligation is sensitive to the stability of
tertiary structure, so this result might reflect the influence of the intracellular
environment on the strength of hairpin ribozyme tertiary interactions. Experiments
are under way to distinguish between these explanations.
Using this simple system, we hope to reveal mechanisms of intracellular RNA
assembly and catalysis that are intrinsic to the RNA sequences and that reflect
general features of the intracellular environment. Insights gleaned from investigation
of intracellular hairpin ribozyme catalysis also will facilitate rational design
of antisense ribozymes for specific and efficient cleavage of RNA targets in
Donahue, C.P., Fedor, M.J. Kinetics of hairpin ribozyme cleavage in
yeast. RNA 3:961, 1997.
Fedor, M.J. Capturing a speeding locomotive. Cell 88:589, 1997.
Fedor, M.J. Ribozymes. Curr. Biol., in press.
Nesbitt, S., Hegg, L.A., Fedor, M.J. An unusual pH-independent and
metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem. Biol. 4:619,