Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.The twister RNA motif was identified by bioinformatic searches and then validated biochemically to be a small self-cleaving ribozyme (1). This recently discovered class of ribozymes is called twister because its conserved secondary structure resembles the ancient Egyptian hieroglyph “twisted flax.” Representatives of the twister ribozyme class are found in all domains of life, but its biological role has yet to be determined. In addition to twister, the small self-cleaving ribozyme family includes the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes (the glmS ribozyme is upsteam of the the glmS gene that codes for the enzyme that catalyzes glucosamine-6-phosphate production) (1–6).The small self-cleaving ribozyme family can be split into two groups based on whether their active site is formed by an irregular helix (hammerhead, hairpin, and VS) or a double pseudoknot (PK) structure (HDV and glmS) (7). The structures of HDV and glmS are known, whereas twister was predicted from representative sequences to use two PKs to form its active site. It was expected that twister would be smaller in size than either HDV or glmS and more comparable in size and complexity to hammerhead (1).The self-cleavage rate constant of twister is estimated to be as rapid as or slightly more rapid than the hammerhead ribozyme. The estimated rate constants (k_obs) for twister is 1,000 per minute, and the experimental k_obs for hammerhead is 870 per minute (1, 8). These two ribozymes are ∼100- to 500-fold faster than other small self-cleaving ribozymes (2–10 per minute under the similar in vitro reaction conditions) (1, 8–10). Twister constructs previously tested exhibited a maximum cleavage rate at 1 mM Mg²⁺ and pH 7.4 (1). However, twister does not require magnesium or other divalent cations for catalysis; thus, magnesium is important only for structure formation (1).Biochemical in-line probing experiments and bioinformatics suggested that the consensus secondary structure of twister contains three to six stems, of which P1, P2, and P4 are required, whereas P0, P3, and P5 are optional; that the RNA can be circularly permutated; and that it contains internal and terminal loops that form two PKs (1, 11). Mutations in any of the highly conserved nucleotides or mutations that disrupt the P1 stem, P2 stem, P4 stem, or the two PKs significantly decrease the catalytic rate (1). Other mutational analysis indicated that several nucleotides are important for self-cleavage, but these nucleotides were not expected to be involved in canonical Watson–Crick (WC) base pairing or displayed any covariation (1).All small self-cleaving ribozymes undergo a specific internal transesterification reaction in which the ribose 2′-oxygen, phosphorus, and 5′-ribose oxygen are aligned for an S_N2-like reaction, yielding products with a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. This single-step reaction is analogous to the reaction catalyzed by RNase A, except that the protein enzyme undergoes a second step to remove the cyclic phosphate (12). There are four general strategies contributing to RNA self-cleavage via internal phosphoester transfer: (i) orientation of the reactive atoms for in-line nucleophilic attack; (ii) neutralization of the negative charge on the nonbridging oxygen atoms of the cleavage site phosphate; (iii) deprotonation of the 2′ oxygen nucleophile; and (iv) neutralization of the developing negative charge on the 5′ leaving group (9). First, all small self-cleaving ribozymes likely use the first strategy for phosphoester transfer, including twister (1, 9). Second, a part of the rate constant enhancement of twister is likely due to the base catalysis because the rate constant has a pH dependency suggesting that the shifts the pK_A of the 2′-hydroxyl group at the cleavage site (1, 9). However, it is unknown whether twister uses the transition-state stabilization and/or general acid strategies (1, 9).Here, we present the 4.1-Å resolution crystal structure of a twister ribozyme sequence from an organism found in the environment that has not been isolated of likely of prokaryotic origin (twister A) and the 3.1-Å resolution X-ray crystal structure of an Orzyza sativa (twister B) twister ribozyme in which the scissile phosphate and nucleotide 5′ to the cleavage site are ordered in the first and disordered in the second. These two crystal structures provide insights into twister’s catalytic mechanism and structural motifs used for formation of its active site. We identify groups that are involved in general base catalysis, transition state stabilization, and provide information about tertiary interactions that form the active site of twister. These structures show variations about how an RNA achieves site-specific self-cleavage and suggest a physical basis for how twister is able to rapidly self-cleave.