Marcello Forconi

Metal Ion-Based RNA Cleavage as a Structural Probe

It is well established that many metal ions accelerate the spontaneous degradation of RNA. This property has been exploited in several ways to garner information about RNA structure, especially in regards to the location of site-specifically bound metal ions, the presence of defined structural motifs, and the occurrence of conformational changes in structured RNAs. In this chapter, we review this information, briefly giving strengths and limitations for each of these approaches. Finally, we provide a general protocol to perform metal ion-mediated cleavage of RNA.

Functional Identification of Ligands for a Catalytic Metal Ion in Group I Introns

Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.

Structure and Function Converge To Identify a Hydrogen Bond in a Group I Ribozyme Active Site

The determination of how enzymes achieve their catalytic power requires an understanding of how structural motifs are used to position functional groups of enzymes and substrates within active sites. The recent explosion of RNA crystal structures provides an extraordinary opportunity to delve deeply into the relationship between ribozyme structure and function. The Tetrahymena group I ribozyme provides an attractive system for such studies because of the wealth of structural information, with ten crystal structures of group I introns solved in the past five years,[1–5] and extensive functional information[6] that enables incisive analysis of the energetics of catalysis.

Tautomerism and Dimerization of Acetamidothiazole Derivatives − UV/Vis and NMR Spectroscopic Investigation

Amido (A)/imido (B) tautomerism has been investigated by UV/Vis and NMR spectroscopic methods for a number of 2-acetamidothiazoles and 2-acetamidobenzothiazoles, without the use of the physical properties of “fixed parents”. The addition of polar substances to solutions of selected compounds in apolar solvents (carbon tetrachloride, dichloromethane) strongly affects the [B]/[A] ratios. Data show that the shift of the tautomeric equilibrium A/B towards the B form has two main causes: (i) increase of the polarity of the medium, and (ii) a base effect on the stabilization of the B form. The experimental ΔH and ΔS values indicate (in agreement with 1H NMR spectroscopic data) that the self-assembly of 2-acetamido derivatives is a very important factor in determining the position of the tautomeric equilibrium.

Tightening of active site interactions en route to the transition state revealed by single-atom substitution in the guanosine-binding site of the Tetrahymena group I ribozyme.

Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNAs structural limitations, but there is little information on the functional and the energetic properties of these inferred networks. We used double mutant cycles and presteady-state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds toward the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.

Use of Phosphorothioates to Identify Sites of Metal-Ion Binding in RNA

Single atom substitutions provide an exceptional opportunity to investigate RNA structure and function. Replacing a phosphoryl oxygen with a sulfur represents one of the most common and powerful single atom substitutions and can be used to determine the sites of metal-ion binding. Using functional assays of ribozyme catalysis, based on pre-steady-state kinetics, it is possible to extend this analysis to the transition state, capturing ligands for catalytic metal ions in this fleeting state. In conjunction with data determined from X-ray crystallography, this technique can provide a picture of the environment surrounding catalytic metal ions in both the ground state and the transition state at atomic resolution. Here, we describe the principles of such analysis, explain limitations of the method, and provide a practical example based on our results with the Tetrahymena group I ribozyme.

Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

Kemp Eliminase Activity of Ketosteroid Isomerase

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

6 How the Group I Intron Works: A Case Study of RNA Structure and Function

In 1968, Leslie Orgel and Francis Crick wrote back-to-back articles in the Journal of Molecular Biology , making the same controversial point: that RNA could, because it could adopt structure (at the time, tRNA was known to adopt its famous “cloverleaf” secondary structure) (Fig. 1), act functionally as a catalyst (Crick 1968; Orgel 1968). Thereby, a solution to the chicken-and-egg problem of the origin of life was proposed. Instead of having to coevolve an information carrier (such as DNA) and a functional macromolecule (such as proteins) to copy information from generation to generation, RNA could have served both roles (Woese 1967; Crick 1968; Orgel 1968). However, the bold suggestions of Orgel and Crick were largely ignored until 1982, when Cech and coworkers discovered the self-splicing activity of the group I intron from Tetrahymena thermophila (Kruger et al. 1982; Cech 1992). The ability of RNA to serve as an information carrier is obvious because it has the same code as DNA and is even used as such in viruses, but RNA’s ability to serve in a capacity analogous to modern-day proteins was not so obvious. Indeed, when this phenomenon was first encountered, it was disbelieved by many and thereafter viewed as mysterious. The difficulty in appreciating RNA as a functional, catalytic molecule stemmed from both the lack of familiarity with RNA structure (crystal structures were available only for tRNA [Robertus et al. 1974; Suddath et al. 1974; Giege et al. 1977; Hingerty et al. 1978; Sussman et al. 1978; Woo et al....