Three metal ions at the active site of the Tetrahymena group I ribozyme

Abstract

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3-oxygen of the oligonucleotide substrate and the 3-and 2-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA. D ivalent metal ions play a critical role in catalysis by many RNA and protein enzymes (e.g., see refs. 1-10). Determining the number of metal ions in an enzymatic active site and delineating their catalytic roles are crucial for elucidating the catalytic mechanisms of these enzymes (e.g., refs. 1-3, 5, 9, and 11-34). This presents a formidable challenge, especially for RNA enzymes, as the metal ions that directly participate in the chemical transformation are bound within a sea of metal ions that coat the charged RNA backbone and facilitate RNA folding ( Fig. 1; e.g., see refs. 32-39).The Tetrahymena group I ribozyme (E) derived from a selfsplicing group I intron catalyzes a reaction that mimics the first step of splicing, in which an exogenous guanosine nucleophile (G) cleaves a specific phosphodiester bond of an oligonucleotide substrate (S; Eq. 1; refs. 40-42).Three metal ion interactions contribute to catalysis by this ribozyme (Fig. 2). These interactions were previously identified by modification of specific substrate atoms that alter metal ion specificity; reactions with sulfur-or nitrogen-substituted substrates were severely compromised in Mg 2ϩ but were stimulated by addition of softer metal ions such as Mn 2ϩ (3,5,7,9). A fundamental question that remains unanswered, however, is whether these interactions are mediated by the same or by distinct metal ions.We report here the development of an approach that combines atomic-level substrate modifications with quantitative analyses to determine the affinity of individual metal ions for an enzymatic active site. These affinities provide a fingerprint for each metal ion, allowing distinct metal ions to be distinguished. Using this approach, we have provided evidence for three distinct metal ions within the active site of the Tetrahymena ribozyme. The results and the approach described herein will allow us to further probe the functional consequences of specific metal ion interactions and the catalytic role of individual metal ions, even within the sea of metal ions bound to RNA. Materials and MethodsMaterials. Ribozyme was prepared by in vitro transcription with T7 RNA polymera...

In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage

“…38 Some large ribozymes use multiple metal ions but without cooperativity. [39][40][41] It was proposed that the VS ribozyme binds four Mg 2+ ions, but the affinity is very low (Kd = 17 mM). 42 We recently reported that the Tm7 DNAzyme employs three Ln 3+ ions cooperatively.…”

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

“…At 2 nM L-16 ScaI ribozyme, most CUCdTAAAAA is unbound (K d ~ 100 nM, data not shown), as suggested from the rate of reaction compared to that for a substrate that is fully bound to ribozyme. Binding of G and UCG to the open complex was determined with 5′-splice site analogs with a 2′-methoxyribose substitution at position -3 (-3m,rSA 5 ,Chart 1) or with 2′-deoxyribose substitutions at all positions other than the cleavage site (-1r,dSA 5 ,Chart 1, (4,35)). …”

“…UCGA was therefore used to probe the reverse of the L-16 ScaI ribozyme reaction without base pairs to the IGS extension formed. 4 UCGA binds to the L-16 ScaI ribozyme with P bound in the closed complex with a dissociation constant of 1050 μM, 12-fold weaker than binding to the L-21 ScaI ribozyme ( Figure 2B and C). This destabilization could arise from interference, direct or indirect, between P9.0 and the IGS extension, as suggested previously and above for binding of UCG (47).…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…However, Basu and Strobel report the rescue of outerspherely coordinated binding sites by Mn 2+ [153]. Since Mg 2+ preferentially binds to phosphoryl oxygens, the most common mutation in this kind of experiment is the exchange of phosphates for phosphorothioates, but also 2'-OH substitution by NH 2 has been used [154].…”

“…Qualitative and quantitative assessment of sequence specific ion binding to DNA Atracts has been undertaken based on the reduced effective charge that can be detected by free solution capillary chromatography [271,272]. Apparent metal affinity constants have been determined from observed rate constants of ribozyme catalysis in many cases (e.g., [154,157,[273][274][275]). An approach based on the gas-phase fragmentation of metal-nucleic acid complexes in ESI-MS (electrospray ionization mass spectrometry) was recently applied to determine binding affinities to the thrombin binding aptamer [276].…”