Crystal structure of Pistol, a class of self-cleaving ribozyme Laura A. Nguyen\"

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Crystal structure of Pistol, a class of self-cleaving ribozyme Laura A. Nguyen", Jimin Wang' and Thomas A. Steitz e' Molecular biochemistry and Bophysia, Yale University, New Haven CT oss20: Howard Hughes Medical Institute, Yale Univeruty, New Haven CT 06520, and Department of Chemistry Yale University, New Haven, CT ois20E107 Edited by Jennifer A Doudna, University of California. Berkeley, CA and approved December 9, 2016 veceived for review luly 2010 can enhance the rates of the intermal transesteriication reaction by Small self-cleaving ribozymes have been discovered in all evolution- ary domains of Me. They catalyze site-specific RNA deavage, and using catalytic strategies, such as deprotonation of the 2-hydromol result they have relevance in gene regulation. Comparative group and neutralising the negative charge on the nonbridging genomic analysis has led to the discovery of a new dass of smal self oxygen of the scissile phosphate or 5-omogen of the cleaved sub- cleaving ribozymes named Pistol. We report the crystal structure of 0, 13-150. We the aystalstructure or Pistol at 29m Aresolution, our Pistol at 2.97A resolution our results suggest that the Pistol ribo- zyme self-cleavage mechanism likely guanine base in the ac- structure reveals the nucleobases that are likely to be involved in the tive site pocket to camy out the phosphoester transfer reaction. The intennal trawesterificatioe reaction of Pistol The structure validates prior biochemical results of the Pistol selfdeavage mechanism and guanine G40 is in dose proximity to serve as the general base for activating the nuceophile deprotonating the 2-hydroxy to ini- further elucidates additionalmechanistic details that camat be easily tiate the reaction (phosphoester transfer. Furthermore,G40 canalso addressed with biochemical analysis (10. The structure shows that establish hydrogen bonding interactions with the nonbridging oxy Paol adopts an overall compactfold stabilized by the Aminor motif ing group suggests that G32 may putatively serve as the general for the stretch of adenines lound in Pitol. sequence conservation acid. The RNA structure of Pistol also contains Aminor interactions The overall fold and cleavage mechaniam of Pistol shares similar which seem to be important to maintain its tertiary structure and features with other self-deaningnbozymes, such as the presence ofa compact fold, our findings expand the repertoire of ribozyme struc- pseudoknot fold and the proposed use of guanosine tures and highlight the conserved evolutionary mechanism used by base, highlighting their comerved evolutionary mechaniam. ribozymes for catalysis. Results and Discussion X-ray crystallography I riboayme I self deavage Structure Determination. The Pistol RNA construct used for crys- intemal transesterification IAminor interaction derived from an extensive comparative genomic analysis of the previously identified environmental sample 27 construct contains two RNA strands annealed together one being out the ma the enzyme strand and the other being a salstrate strand (Fig I) lution of complex protein enzymes 20. Ribozymes are non- The substrate strand contains the Pistol sel deavage site, coding RNA that carry out catalytic activities. Unlike protein which is positioned between guanosine 0 (G10) and uridine 11 enzymes, only a handful of riboaymes have known biological EUil) (Fig. LB To trap the Pistol riboayme functions Their biological functions range from regulating gene state for generated a Pistol RNA substrate riboswitches and performing peptidyltransfer removing intron sequences in genes (eg- self-splicing Group l intron ribozymes) 2-9) The biological functions and mechanism of these ribozymes have been discovered through structural and biochemical studies. Based on the "RNA world" theory, ribozymes likely camied out Currently, the classes of selfdeaving ribozymes consist of Ham- biochemical reactions long before organisms evolved to use merhead Hairpin, Hepatitis Delta Virus ribozyme (IDN). Varkud protein enzymes as biocatalysts. The continued discovery of new structures for small selfdeaving riboaymes has shed light on Satellite (VS0. glms ribozyme, Twister, Twister sister, Hatchet, and Pistol (10) These classes differ based on their size, structure, and conserved mechanisms in evolution, such as acid-base catalysis for self-deavage reaction. Here, we present the oystal structure cleavage mechanism. Known for their site-specific cleavage, ribo- zymes with defined biological function include the Hammerhead, of a newly discovered dass of self-deaving riboaymes called VS, and HDV, which all participate Pistol and how it likely uses the phosphoester transfer mecha in rolling circle replication. nism for self deavage. The results presented here suggest that whereas the glms ribozyme functions in controlling gene expression Pistol uses an evolutionarily conserved deavage mechanism that (11, 12 However, the biological function of a vat majority of the is like other self deaving riborymes, such as Twister, Hammer- different self-deaving ribozymes remains to be explored. head, Hairpin, and Hepatitis Delta Virus ribozymes Through comparative genome analysis, there have been three newly identified danses of self-cleaving ribozymes called Twister sider, Hatchet, and Pistol G. Biochemical analysis reveals that Pistol can use a variety of divalent metal ions to carry out a com- plete site -specific self-cleaving reaction, whereas utilization of monovalent cations results in modest cleavage rates (3 100 The rate of Pistol selfdeavage has been estimated to be 10 min under physiological conditions and >100 min under optimal magnesium and pH conditions (0) Pistol self deavage is via an internal transesterification reaction, in which the substrate RNA of Pistol G102 -hydroaylon the nitose makes anuckophilic attack on adjacent 3-phosphate Sellceaving ribozymes January 31.2017 no.

Explanation / Answer

A very significant small molecular ribozyme, with relevance in gene regulation, due to its property of site specific self-cleavage have been discovered, which has been termed as PISTOL. Authors have reported the crystal structure of molecule and further added that the mechanism of self-cleavage use guanine base, especially G40, which serves as a general base for nucleophilic attack by deprotonating the 2-OH, present at the active site pocket. However, the exact biochemical mechanism is yet to come out. These ribozymes use many divalent metal ions to complete the process of cleavage. The discovery of self-cleaving ribozymes could be act as a source to explain the conserved mechanism in evolution.

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