User:Xikun Liu/sandbox

Discovery

The twister sister ribozyme (TS) was discovered by Beaker et al, in 2015 through a bioinformatic search. Based on the fact that many twister and hammerhead ribozymes are located near to each other and near certain protein-coding genes, Beaker et al, searched for the conserved RNA structures near those genetic elements and tested the self-cleaving activity of those RNA motifs. Three RNA motifs show self-cleaving activity. One of the three is remotely similar to twister so it’s named twister sister. The other two are named pistol and hatchet.

Structure

The crystal structure of the pre-catalytic state of a three-way junctional twister sister ribozyme was reported by Lilley et al. It is composed of two co-axial stacked helical sections connected with a three-way junction and two tertiary contacts. The active site, a scissile phosphate is located in a loop with quasihelical character and it’s in one coaxial base-stacked helix. Five divalent metal ions are coordinate to RNA ligands, one of which is directly bound to C 54 O2’ near the scissile phosphate and exchange inner sphere water molecules with the RNA ligands.

The crystal structure of a four-way junctional twister sister ribozyme is different from the three-way junctional one in terms of long-range interaction and active site structure according to Ren et al. The active site of a four-way junctional twister sister is splayed-apart with an interaction between guanine and scissile phosphate. Besides, there are seven divalent metal ions in this ribozyme.

Catalytic Mechanism

Generally, nucleolytic ribozyme cleave a specific phosphodiester linkage by SN 2 mechanism. The O2 acts as a nucleophile to attack the adjacent P, with O5’ as a leaving group. The catalytic products are a cyclic 2’,3’ phosphate and a 5’-hydroxyl

The catalytic activity of twister sister increases with pH and depends on divalent metal ion. The cleavage speed increases 10 fold with each increase in pH unit and reach plateau near pH 7, which indicates that the 2-hydroxyl group of cytidine near the active site is fully deprotonated at pH 7 in the ribozyme. However, the structure basis for the catalytic activity is still under investigation.

Lilley’s research group claims that the three-way junctional twister sister is a metalloenzyme. The inner sphere water of a divalent metal ion bound to C 54 O2’ acts as a general base to deprotonate the 2-hydroxyl group, making it a stronger nucleophile, but the general acid which can stabilize the oxyanion leaving group remains unknown. This mechanism is supported by the exponential correlation between catalytic activity and the pKa of hydrated metal ion.

For four-way junctional twister sister, Ren and coworkers find out that guanine with an amino group is likely to play a role in the catalysis because G5 mutations result in very low catalytic activity. However, it remains unclear whether guanine directly participate in the catalysis because it’s not absolutely conserved. Recently, Micura et al, point out that the formation of pesodoknot for four-way junctional TS is highly Mg2+ dependent by conducting SHAPE (Selective-2′ -Hydroxyl Acylation analyzed by Primer Extension) experiment.

So far, we only know the pre-catalytic conformation of twister sister ribozymes. The structure determination of transition state is needed to explain the relationship between twister ribozyme and twister sister ribozyme as well as the structure differences of active site between the three-way and four-way junctional twister sister.