Structures Of Twister-sister And Hatchet Self-cleaving Ribozymes And Implications Of Cleavage Mechanism

Ribozymes are non-coding RNAs that can catalyze specific biochemical reactions without the assistance of proteins,involved in a variety of vital cellular reactions,including t RNA processing,intron splicing,protein synthesis,and so on.According to catalytic mechanisms,ribozymes can be divided into RNA metalloenzymes and self-cleaving ribozymes.Previous studies have shown that self-cleaving ribozymes adopt a general acid-base catalytic mechanism to perform site-specific phosphodiester bond cleavage.Until now,only ten families of self-cleaving ribozymes have been discovered,termed hammerhead,HDV,VS,hairpin,glm S,twister,pistol,twister-sister,hatchet and hovlinc.Based on the catalytic properties of self-cleaving ribozymes,researchers have developed various artificial systems for applications in in vitro transcription of RNA,in vivo regulation of gene expression,gene editing,etc.The determination of the tertiary structure of self-cleaving ribozymes can promote progress on the catalytic mechanisms and their applications.In this paper,we solved the three-dimensional structures of twister-sister and hatchet self-cleaving ribozymes by X-ray crystallography and made discussions on their catalytic mechanisms.We determined the crystal structure of a four-way junctional twister-sister self-cleaving ribozyme.A d C62 was introduced into the cleavage site to prevent self-cleavage of the ribozyme.Eleven conserved spatially separated loop nucleotides are brought into close proximity at the ribozyme core through long-range interactions.The C62-A63 step at the cleavage site adopts a splayed-apart orientation,with A63 is directed inwards and anchored by stacking and hydrogen-bonding interactions,whereas flexible C62 directed outwards.A guanine G5 at the active site forms interactions with the non-bridging oxygen of the scissile phosphate,contributes to the cleavage chemistry.The above feature is also found in twister ribozyme.This G5 has a vital role in the cleavage chemistry,although its direct participation in catalysis remains to be proven.Several hydrated Mg²⁺are found around the cleavage site and form hydrogen bond networks.The modeled 2’-OH of C62is positioned for hydrogen bonding to an inner-sphere water of hydrated Mg²⁺labeled M2,suggesting a potential general base role in catalysis.The precatalytic structure of twister-sister ribozyme reveals structural characteristics of overall folding and catalytic pocket,which provides a basis for selecting mutation sites for studying the cleavage chemistry,and further promotes the study of catalytic strategies and mechanisms.Hatchet ribozyme is the only one featured with a very 5’end cleavage site among all three newly found self-cleaving ribozymes.In this paper,we showed the 2.1-（?）crystal structure of the 3’product of hatchet ribozyme.Since all the conserved sequence and main secondary structure elements are located in the 3’product,the above structure can represent the overall structure of the full-length hatchet ribozyme.The overall structure of 3’product of hatchet ribozyme adopts a compact pseudosymmetric dimeric scaffold,with each monomer stabilized by long-range interactions between L1 and L3,as well as highly conserved nucleotides brought into close proximity of the scissile phosphate.The catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5’nucleoside.The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate and a series of hydrogen bonds are formed between highly conserved nucleotides and the modeled scissile phosphate and its flanking 5’nucleoside.We identify a guanine lining the catalytic pocket positioned to contribute to the cleavage chemistry and served as the potential general base.The tertiary structure of 3’product of hatchet ribozyme provides the overall view of intricate tertiary fold and key residues lining the catalytic pocket.Therefore,this study provides a structure-based platform to obtain full-length structure and understanding of the catalytic mechanism of hatchet ribozyme.