The Skaggs Institute
for Chemical Biology
Scientific Report 2005
Intracellular RNA Folding and
M.J. Fedor, E.M. Calderon,
C.P. Da Costa, J.W. Cottrell, J.W. Harger, Y.I. Kuzmin, E.M. Mahen
perform essential roles in the expression and propagation of genetic information, providing
potential targets and reagents for therapeutic intervention. RNAs adopt precise 3-dimensional
structures to carry out their biological functions, and sometimes a single RNA must adopt several
distinct structures during the course of a reaction. Despite extensive studies of RNA folding
in vitro, the multistep nature of most RNA-mediated reactions and the complexity of the intracellular
environment limit the accuracy with which simple in vitro reactions can recapitulate RNA folding
in vivo. We are studying ribozyme self-cleavage reactions in yeast to learn how principles of RNA
folding established through in vitro experiments translate to the behavior of RNAs in a biological
context. RNA self-cleavage gives a clear, quantitative signal that a catalytic RNA has assumed
its functional structure, making ribozymes particularly useful models for RNA folding studies.
To investigate how RNAs can adopt specific
functional structures despite the capacity to form alternative nonfunctional structures with
similar stabilities, we designed hairpin ribozymes with the potential to form defined stem-loop
structures that are incompatible with assembly of a functional ribozyme (Fig. 1).
|Fig. 1. Hairpin ribozyme sequences with the potential to form
either functional ribozymes or nonfunctional stem-loop structures. The secondary structure
of an active ribozyme contains 4 essential base-paired helices, H1 through H4. The functional
ribozyme structure contains an active site that catalyzes self-cleavage of the phosphodiester
bond indicated by the arrows. A, The upstream AltH1 ribozyme variant has a sequence added to the
5′ end (bold letters) that can pair with 10 bases at the 5′ end of the ribozyme to form an Alt5′ H1
stem loop that is incompatible with assembly of an active ribozyme. If secondary structures assemble
sequentially during RNA synthesis, Alt5′ H1 would form before all of the sequences needed to form the essential H1 helix of the ribozyme have
been synthesized, and the Alt5′ H1 insert would inhibit assembly of an active ribozyme. B, The downstream AltH1 variant has a sequence
added to the 3′ end of the ribozyme (bold letters) that can pair with 10 bases at the 3′
end to form an Alt3′ H1 stem loop that is incompatible with assembly of a functional ribozyme. If secondary structures assemble
sequentially, the complete ribozyme sequence would be able to assemble into an active ribozyme
before the sequence needed to form Alt3′ H1 has been synthesized, so the Alt3′ H1
insert would not interfere with ribozyme self-cleavage activity.
In one variant,
a complementary insert located upstream of the ribozyme can form 10 base pairs with the 5′
end of the ribozyme sequence to create a stem loop, Alt5′ H1,
that is incompatible with assembly of the essential H1 helix of the ribozyme. In another variant,
an insert located downstream of the ribozyme can form 10 base pairs with 3′
terminal ribozyme nucleotides to form a stem loop, Alt3′ H1,
that also is incompatible with H1 helix assembly. Because formation of either AltH1 stem loop blocks
assembly of a functional ribozyme, we can monitor partitioning between functional ribozymes
and nonfunctional stem loops via inhibition of self-cleavage.
Ribozymes with upstream or downstream
inserts had very different self-cleavage activity in cotranscriptional assembly reactions
in vitro, consistent with a sequential folding mechanism. A ribozyme with an upstream insert self-cleaved
very slowly, suggesting that a stem loop that sequesters the upstream part of H1 blocks ribozyme
assembly. In contrast, ribozymes retained significant activity when a complete ribozyme was
synthesized before a downstream insert was made in vitro. These different effects of upstream
and downstream inserts suggest that structures assemble sequentially in vitro so that upstream
structures that can assemble first dominate the folding pathway.
Partitioning between functional ribozymes
and nonfunctional stem loops did not show the same polarity in yeast; a downstream insert blocked
ribozyme assembly completely even though the entire ribozyme was synthesized first. This evidence
that a downstream stem loop can compete effectively with ribozyme assembly suggests that some
feature of the intracellular environment affects folding in some way that simple in vitro reactions
do not recapitulate. In vivo, Alt3′ H1
and H1 might interconvert rapidly, allowing the most thermodynamically stable structure to predominate
at equilibrium (Fig. 2A). However, this rapid exchange model is inconsistent with
the high thermodynamic stability that is characteristic of helices with 8 or more base pairs, which
are expected to remain stable for days.
|Fig. 2. Models for RNA folding in vivo. A, In a rapid-exchange model, secondary structures that form during transcription
can interconvert at a rate that might be accelerated through the action of RNA chaperone proteins
(shaded spheres). B, In a delayed-folding model, assembly of secondary structure does not occur
during RNA synthesis, perhaps because nascent transcripts associate with RNA-binding proteins
(shaded ellipses). Stable structures might form only after an entire folding domain has been released
from bound proteins. RNAP indicates RNA polymerase.
If exchange occurs within minutes during
RNA self-cleavage in vivo, some intracellular factors, such as RNA chaperones, must accelerate
interconversion between H1 and AltH1. However, no nonspecific RNA chaperone activity has ever
been detected in vivo. Alternatively, assembly might be delayed, perhaps by the reversible association
of proteins with nascent transcripts (Fig. 2B). In a delayed folding model, dissociation
of proteins might release upstream and downstream RNA sequences to fold simultaneously so that
structures that assemble quickly from contiguous sequences might dominate the folding pathway.
Our ongoing experiments are designed to
distinguish between rapid exchange and delayed folding models.
Fedor, M.J., Williamson, J.R.
The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6:399, 2005.
Kuzmin, Y.I., Da Costa, C.P., Cottrell,
J.W., Fedor, M.J. Role of an active site adenine in hairpin
ribozyme catalysis. J. Mol. Biol. 349:989, 2005.
Mahen, E.M., Harger, J.H., Calderon,
E.M., Fedor, M.J. Kinetics and thermodynamics make different
contributions to RNA folding in vitro and in yeast. Mol. Cell 19:27, 2005.