The Skaggs Institute
for Chemical Biology

Scientific Report 2005

Intracellular RNA Folding and Catalysis

M.J. Fedor, E.M. Calderon, C.P. Da Costa, J.W. Cottrell, J.W. Harger, Y.I. Kuzmin, E.M. Mahen

RNAs 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.

Publications

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.