Identification and Characterization of Catalytic Metal Ions Within RNA
The Tetrahymena ribozyme is a metalloenzyme that catalyzes cleavage of oligonucleotide substrates via phosphoryl transfer, analogous to the first step in group I intron self-splicing. Divalent metal ions, which are essential for RNA folding and function, play an intricate role in catalysis by this ribozyme. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme.
In collaboration with Dan Herschlag and his colleagues (Stanford University), we have used atomic-level substrate modifications coupled with quantitative mechanistic analyses to obtain evidence for a novel assembly of catalytic interactions involving three metal ions. In the transition state, the 3'-oxygen leaving group interacts with MA, the nucleophilic 3'-OH of G interacts with MB, the 2'-OH of G interacts with MC, and the nonbridging pro-SP oxygen of the scissile phosphate interacts with both MA and MC. Presumably, MA stabilizes the developing negative charge on the leaving group and together with MC destabilizes the developing negative charge on the nonbridging oxygen atom in the transition state; MB may enhance the nucleophilicity of the 3'-OH of G, perhaps by lowering its pKa. One or more of these metal ions also may position and align the reactive groups.
Recently we have shifted our focus toward identifying the functional groups within the RNA that bind to these catalytic metal ions. We have combined the approach outlined above with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a general strategy for defining ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified a specific ligand for MC. Functional identification of metal ion ligands within enzyme active sites provides critical information for evaluating models based on structural data and offers powerful insights into strategies for metalloenzyme catalysis.
Energetics and Cooperativity of Metal-Ligand Interactions
Metal ion–ligand interactions in RNA have been identified at atomic resolution by x-ray crystallography but not analyzed thermodynamically. Conversely, RNA tertiary interactions have been quantified energetically, but it was unclear whether metal-ligand interactions were involved, as these interactions were not viewed at atomic resolution. We have been investigating the thermodynamics of metal-ligand interactions observed in the x-ray crystal structure of the P4–P6 folding domain of Tetrahymena group I intron. These studies will allow a better understanding of the thermodynamic contributions that stabilize folded RNA structures.
Exploring RNA's 2'-Hydroxyl Group
RNA's 2'-hydroxyl groups provide critical contributions to structure and function. We have developed experimental strategies that reveal the chemical basis of 2'- hydroxyl contributions from tertiary interactions (via H-bonding or metal ion coordination), from indirect factors (via electrostatic effects, spatial occupancy, sugar conformation, and inductive effects), and from solvent interactions. The nucleoside analogs span a broad range of chemical diversity and allow use of quantitative structure activity relationships (QSARs) in the exploration of RNA biology. We employed these strategies to investigate the spliced-exons reopening (SER) reaction of the group II intron. Our results suggest that group II introns invade splice junctions containing ribose at the cleavage site faster than those containing deoxyribose because the cleavage site 2'-hydroxyl mediates an interaction with an important water molecule.
An atomic mutation cycle. We have used a subset of these analogs to develop an atomic mutation cycle that reveals whether a hydroxyl group imparts a functional contribution via hydrogen-bond donation. The cycle describes the energetic effects from three atomic mutations (2'-OCH3, 2'-NH2, and 2'-NHCH3) relative to the ribonucleotide (2'-OH) on an RNA-mediated process. This approach bears some similarity to double-mutant cycles used in protein analysis except that mutations occur at the level of atoms rather than residues: the –O- and –H atoms of the 2'-hydroxyl group are mutated to –NH- and –CH3, respectively. When the cost of 2'-methoxynucleotide substitution exceeds that for methyl-group installation, this analysis indicates that the hydrogen atom of the 2'-hydroxyl group contributes significantly to function, presumably by donating a hydrogen bond. In contrast, when the cost of 2'-methoxy substitution matches that measured for methyl-group installation, the cycle provides no evidence that the 2'-hydroxyl group donates a functionally important hydrogen bond.
A packing-density metric for exploring the interior of folded RNA molecules. RNA molecules adopt complex tertiary architectures that contain expansive packing interfaces. Within these interfaces, atoms acquire distinct packing environments that uniquely specify an RNA fold. To define the packing environment surrounding individual 2'-hydroxyl groups, we have synthesized a series of 2'-modified nucleotides (2'-chloro, 2'-methyl, and 2'-mercaptonucleotides) that span a narrow range of molecular volume. We incorporated these analogs transcriptionally into the structurally and biochemically well-defined P4–P6 domain and determined the sites at which the analogs interfere with folding. Our results showed that the frequency of analog interference increases with molecular volume and that the residues in the domain exhibit a volume threshold above which RNA folding becomes destabilized. The resulting interference profiles reflect the spatial environment surrounding the 2'-hydroxyl groups, correlating with the number and closeness of neighboring atoms so as to define a packing-density metric. This metric may be used as a "fingerprint" to identify similar motifs that occur in other biological RNAs whose structures have not been determined.
A Mechanistic Probe for General Acid/Base Catalysis by RNA
There is evidence that some naturally occurring RNA enzymes (the hepatitis delta virus [HDV] ribozyme, the hairpin ribozyme, and the ribosome) use direct functional group participation rather than divalent metal ions to facilitate catalysis. However, in contrast to the approaches described above to test for metal ion catalysis, there exists no simple approach to test directly for general acid/base catalysis. We have been developing such an approach in the context of the HDV ribozyme, which catalyzes RNA strand scission by facilitating attack of the 2'-OH on the adjacent phosphodiester. An active-site cytidine, protonated at N3, is proposed to act as a general acid catalyst, donating a proton to the leaving group in the transition state. We have been testing this proposal by synthesizing a modified substrate in which a sulfur atom replaces the oxygen leaving group. If a general acid donates a hydrogen bond to the leaving group in the transition state, then in the context of a sulfur leaving group, mutation of the general acid residue would no longer have a severely deleterious effect on catalysis, as sulfur possesses an inherently greater leaving and weaker H-bond–accepting ability than oxygen. In contrast, mutations at residues other than the one acting as the general acid would affect the reactions of both substrates similarly. If successful, this approach will provide direct functional evidence for general acid catalysis.
