1. Mechanisms of RNA Assembly and Catlysis

2. Intracellular RNA Folding and Catalysis

Mechanisms of RNA Assembly and Catalysis

Our work aims to generate basic insights into catalysis by RNA and RNA-protein enzymes, RNA folding, and RNA interactions with small molecules. In addition to contributing basic knowledge of RNA structure and function in normal growth and development, results of our studies provide a framework for developing technical and therapeutic applications involving RNAs as targets and reagents.

Apart from the ribosome, which catalyzes peptidyl transfer, the naturally occurring ribozymes catalyze phosphate-group transfer. The small RNA enzymes that we study catalyze reversible phosphodiester cleavage reactions that generate 5' hydroxyl and 2',3'-cyclic phosphate termini (Fig.1).

Possible strategies for catalysis of phosphoryl transfer reactions include: aligning reactive groups in an optimal orientation for an in-line attack mechanism; general acid base catalysis of proton transfer to activate nucleophilic oxygens or to stabilize oxyanion leaving groups; electrostatic stabilization of negative charge that accumulates in the transition state; or destabilizing the ground state. Our goal is to understand which of these catalytic strategies RNA enzymes use. In contrast to the chemical versatility of the amino acid side chains that comprise the active sites of protein enzymes, just four nucleotides are available for the construction of ribozyme active sites. Nucleotides are well suited to faithful storage and transmission of genetic information through complementary base pairing, but they are not particularly adept at catalytic chemistry. Protonation and deprotonation of nucleotides occur at high or low pH extremes, which would make it difficult to mediate general acid or base catalysis at neutral pH. No positively charged nucleotide functional groups are expected to be available at neutral pH to function as Lewis acids to activate a nucleophile, or stabilize an electronegative transition state or an oxyanion leaving group.

Recent high-resolution structures of self-cleaving RNAs lay the groundwork for experiments to probe fundamental questions about how RNA enzymes use their functional groups for catalysis. Like all enzymes, hairpin ribozymes combine several strategies to achieve catalytic rate enhancement. One important strategy that is apparent from the crystal structures is the alignment of nucleophilic and leaving group oxygens in the optimal orientation for an in-line SN2-type nucleophilic attack. The structure of the hairpin ribozyme active site places G8, A9, A10, and A38 nucleobases near the reactive phosphate. G8 and A38 occupy positions reminiscent of two histidine residues in the active site of ribonuclease A, a protein enzyme, which catalyzes the same reaction. Histidine residues perform general acid base catalysis during ribonuclease A catalysis, so the similarity between hairpin ribozyme and ribonuclease A active sites raised the possibility that G8 and A38 nucleobases might perform similar functions as histidine residues. Hairpin ribozyme activity increases with increasing pH, consistent with the notion that activity depends on the availability of the deprotonated form of G8 to accept a proton from the 2' hydroxyl nucleophile as predicted by the general acid base catalysis model. To test this model, we replaced G8 with an abasic residue, a substitution that eliminates the nucleobase but leaves the phosphodiester backbone intact. However, this abasic variant displays the same pH dependence as an unmodified ribozyme, arguing that the pH transition does not involve G8. An abasic substitution of A38, on the other hand, does eliminate the pH-dependent transition in activity, implicating A38 in a catalytically important deprotonation.

These and other results are consistent with two models of the hairpin ribozyme catalytic mechanism in which A38 contributes either general acid base catalysis (Fig. 2A) or electrostatic stabilization of negative charge that develops in the transition state as five electronegative oxygen atoms from transient bonds with phosphorus (Fig. 2B) and G8 donates hydrogen bonds to stabilize the transition state electrostatically.

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Intracellular RNA Folding and Catalysis

RNAs perform essential roles in the expression and propagation of genetic information, providing potential targets and reagents for therapeutic intervention. RNAs adopt precise three-dimensional structures to carry out their biological functions and sometimes a single RNA must adopt several distinct structures over 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). In one variant, a complementary insert located upstream of the ribozyme can form ten 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 a second variant, an insert located downstream of the ribozyme can form ten base pairs with 3' terminal ribozyme nucleotides to form a stem loop, Alt3'H1, that also is incompatible with H1 helix assembly. Since formation of either AltH1 stem loop blocks assembly of a functional ribozyme, we can monitor partitioning between functional ribozymes and nonfunctional stem loops through inhibition of self-cleavage.

Ribozymes with upstream or downstream inserts displayed very different self-cleavage activity in co-transcriptional 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 eight or more base pairs, which are expected to remain stable for days. 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 assembly quickly from contiguous sequences might dominate the folding pathway. Our ongoing experiments are designed to distinguish between rapid exchange and delayed folding models.

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