Biochemical analysis of hatchet self-cleaving ribozymes

Abstract

Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg 2+ to promote RNA strand scission with a maximum rate constant of ∼4 min −1 . As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2 ′ -oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

“…The mechanism of self-cleavage of the in vitro selected leadzyme, nucleophilic attack of a 2 ′ -hydroxyl on the adjoining phosphate with departure of the 5 ′ -oxygen, is common to the prominent Mg 2+ -activated family of selfcleaving ribozymes that includes the paradigmatic hammerhead, hairpin, and hepatitis delta motifs (Esteban et al 1997;Fedor and Williamson 2005;Cochrane and Strobel 2008) as well as several more recently discovered species (Roth et al 2014;Harris et al 2015;Li et al 2015;Weinberg et al 2015). The proper alignment of these groups for facile "in-line" attack is a substantial contributor to the catalytic power of these motifs (Soukup and Breaker 1999;Emilsson et al 2003).…”

Numerous small hammerhead ribozyme variants associated with Penelope-like retrotransposons cleave RNA as dimers

“…The relative abundance of self-cleaving RNA classes emphasizes their mysterious roles in biology. The continuing discovery of novel self-cleaving ribozyme motifs [2][3][4] as well as variants of long standing self-cleaving ribozymes such as the hammerhead class will give us greater opportunity to decipher their functions. These discoveries also highlight the fact that modern organisms continue to make extensive use of these catalytic RNAs.…”

“…Of the 15 known classes of natural ribozymes, 9 catalyze RNA self-cleavage by a phosphoester transfer reaction. [1][2][3][4] This reaction involves the nucleophilic attack by a 2 0 -oxygen atom of ribose on the adjacent phosphorus center, which leads to departure of the neighboring 5 0 -oxygen atom and splitting of the RNA phosphodiester backbone. 1,5 Hammerhead ribozymes were the first of these 9 classes to be discovered, [6][7][8] which is not surprising given that members of this ribozyme class are the most common of all natural self-cleaving RNAs.…”

Mechanistic Debris Generated by Twister Ribozymes

“…Coming to agreement on the catalytic strategies used by twister cannot come too soon, as additional data on the structures and mechanisms of the newest self-cleaving ribozyme classes called twister sister, 26 hatchet 27 and pistol 28 are already beginning to be published. 29,30 Moreover, it is almost certain that other natural self-cleaving ribozyme classes await discovery.…”