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Fedor Lab Research

Understanding mechanisms of RNA catalysis remains an intriguing challenge, one that has grown in significance since the recent demonstration that the ribosome is an RNA enzyme. Our research focuses on one catalytic RNA, the hairpin ribozyme, which cuts and rejoins its RNA substrates. Unlike other ribozymes that recruit metal cation cofactors to perform catalytic chemistry, the hairpin ribozyme uses functional groups within the RNA itself to carry out catalysis. We use enzymological, structural and biochemical methods to learn how specific functional groups in the hairpin active site contribute to catalysis. RNA catalysis provides a sensitive, quantitative signal that an RNA has assumed its functional structure making ribozymes, particularly fruitful systems for RNA structure-function studies. Quantitative enzymological studies of ribozymes have led to remarkably detailed insights into the structural transitions that comprise RNA assembly and reaction pathways and defined how interactions with other molecules influence these pathways in vitro. However, the complexity of biological reactions and the lack of information about the intracellular environment limit the accuracy with which in vitro reactions can recapitulate biology. We have devised a way to quantify ribozyme reaction kinetics in yeast in order to learn how principles of RNA folding and catalysis that have been revealed through in vitro studies relate to the behavior of RNAs in living cells. Insights gained from our studies will facilitate the design of ribozymes for therapeutic applications and shed light on more complex RNA-mediated reactions in gene expression.

An active-site guanine participates in glmS ribozyme catalysis in its protonated state

Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2'-OH nucleophile. Apparent pK(a) values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK(a) values obtained from functional assays and microscopic pK(a) values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.

Viladoms Figure

Sequence, structure, and function of the glmS ribozyme. (a) Proposed acid_base mechanism for the self-cleavage reaction catalyzed by the G33 and GlcN6P. (b) Model of the active-site structure showing the scissile phosphate and important guanine residues (based on the X-ray structure of the precleavage state of B. anthracis glmS with bound GlcN6P and 20-OMeA-1, PDB entry 2NZ4, with G8 replaced by 8azaG8 and the methyl group removed from the 20-O of A-1 using PyMol). (c) Sequence and secondary structure of the glmS ribozyme from B. anthracis. G28c is a circularly permuted variant used for kinetic and fluorescence experiments. A-1 and G1 (red) were mutated to C in the inactive ribozyme (G28cm).

 

The glmS Riboswitch Integrates Signals from Activating and Inhibitory Metabolites In Vivo

The glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes. It is also a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS. The ligand concentration dependence of intracellular self-cleavage kinetics was measured for the first time in a yeast model system and unexpectedly revealed that this riboswitch is subject to inhibition as well as activation by hexose metabolites. Reporter gene experiments in Bacillus subtilis confirmed that this riboswitch integrates positive and negative chemical signals in its natural biological context. Contrary to the conventional view that a riboswitch responds to just a single cognate metabolite, our new model proposes that a single riboswitch integrates information from an array of chemical signals to modulate gene expression based on the overall metabolic state of the cell.

 

Watson Figure

Model for riboswitch regulation of GlmS gene expression. Riboswitch cleavage activity integrates information about the metabolic state of the cell by responding to the concentrations and affinities of an array of chemical signals. In this model, hexoses increase glmS mRNA abundance and upregulate GlmS expression by inhibiting riboswitch cleavage (green line), while aminohexoses decrease glmS mRNA abundance and downregulate GlmS expression by activating riboswitch cleavage (red line). The dashed box illustrates the former model that a single cognate ligand, GlcN6P, activates the riboswitch to down-regulate GlmS expression.

 

The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pKa values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pKa for ionization of 8azaA at position 38 with the apparent pKa for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pKa values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.

Cottrell Figure

A, structure of the Hp Rz active site with a vanadate mimic of the transition state prepared from the coordinates (Protein Data Bank code 1M5O) of Rupert et al. B, general acid-base model of the reversible phosphodiester cleavage reaction catalyzed by the Hp Rz.

mRNA Secondary Structures Fold Sequentially But Exchange Rapidly In Vivo

RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts.

Figure 3

Chimeric RNAs contain sequences with the potential to form self-cleaving ribozymes or nonfunctional structures, depending on the relative contributions of kinetics and thermodynamics to RNA folding outcomes. The secondary structure of this self-cleaving ribozyme contains an essential base-paired helix, called H1, with 14 base pairs. a. A downstream insert (green) is complementary to the adjacent ribozyme sequence and has the potential to form an alternative helix with 12 base pairs, called 3'AltH1. During co-transcriptional RNA assembly in vitro and in vivo, the H1 structure that forms first resists competition from the downstream 3'AltH1 and allows assembly of a functional, self-cleaving ribozyme. b. In a second ribozyme variant, an upstream insert (red) is complementary to the ribozyme sequence and can form a helix with 12 base pairs, called 5'AltH1, which is incompatible with H1 assembly. Although the 5'AltH1 helix has lower thermodynamic stability than H1, 5'AltH1 can form first during transcription to create a "kinetic trap" that blocks subsequent assembly of a functional ribozyme both in vitro and in vivo