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Crystal Structure of Pistol, a Class of Self-Cleaving Ribozyme

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Crystal Structure of Pistol, a Class of Self-Cleaving Ribozyme Crystal structure of Pistol, a class of self-cleaving ribozyme Laura A. Nguyena, Jimin Wanga,1, and Thomas A. Steitza,b,c,1 aDepartment of Molecular Biochemistry and Biophysics, Yale University, New Haven, CT 06520; bHoward Hughes Medical Institute, Yale University, New Haven, CT 06520; and cDepartment of Chemistry, Yale University, New Haven, CT 06520-8107 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved December 19, 2016 (received for review July 8, 2016) Small self-cleaving ribozymes have been discovered in all evolution- can enhance the rates of the internal transesterification reaction by ary domains of life. They can catalyze site-specific RNA cleavage, and using catalytic strategies, such as deprotonation of the 2′-hydroxyl as a result, they have relevance in gene regulation. Comparative group and neutralizing the negative charge on the nonbridging genomic analysis has led to the discovery of a new class of small self- oxygen of the scissile phosphate or 5′-oxygen of the cleaved sub- cleaving ribozymes named Pistol. We report the crystal structure of strate (10, 13–15). Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribo- We report the crystal structure of Pistol at 2.97-Å resolution. Our zyme self-cleavage mechanism likely uses a guanine base in the ac- structure reveals the nucleobases that are likely to be involved in the tive site pocket to carry out the phosphoester transfer reaction. The internal transesterification reaction of Pistol. The structure validates guanine G40 is in close proximity to serve as the general base for prior biochemical results of the Pistol self-cleavage mechanism and activating the nucleophile by deprotonating the 2′-hydroxyl to ini- further elucidates additional mechanistic details that cannot be easily tiate the reaction (phosphoester transfer). Furthermore, G40 can also addressed with biochemical analysis (10). The structure shows that establish hydrogen bonding interactions with the nonbridging oxy- Pistol adopts an overall compact fold stabilized by the A-minor motif gen of the scissile phosphate. The proximity of G32 to the O5′ leav- commonly found in many RNA structures, which explains the high ing group suggests that G32 may putatively serve as the general sequence conservation for the stretch of adenines found in Pistol. acid. The RNA structure of Pistol also contains A-minor interactions, The overall fold and cleavage mechanism of Pistol shares similar which seem to be important to maintain its tertiary structure and features with other self-cleaving ribozymes, such as the presence of a compact fold. Our findings expand the repertoire of ribozyme struc- pseudoknot fold and the proposed use of guanosine as a general tures and highlight the conserved evolutionary mechanism used by base, highlighting their conserved evolutionary mechanism. ribozymes for catalysis. Results and Discussion X-ray crystallography | ribozyme | self-cleavage | Structure Determination. The Pistol RNA construct used for crys- internal transesterification | A-minor interaction tallization was derived from an extensive comparative genomic analysis of the previously identified environmental sample 27, he “RNA world” hypothesis speculates that RNA carried likely of bacterial origin (3, 16). The bimolecular Pistol RNA Tout the majority of biochemical reactions before the evo- construct contains two RNA strands annealed together: one being lution of complex protein enzymes (1, 2). Ribozymes are non- the enzyme strand and the other being a substrate strand (Fig. 1) coding RNA that carry out catalytic activities. Unlike protein (3). The substrate strand contains the Pistol self-cleavage site, enzymes, only a handful of ribozymes have known biological which is positioned between guanosine 10 (G10) and uridine 11 B functions. Their biological functions range from regulating gene (U11) (Fig. 1 ). To trap the Pistol ribozyme in its precatalytic expression (e.g., riboswitches) and performing peptidyl-transfer state for crystallization, we generated a Pistol RNA substrate reactions (e.g., ribosome) to removing intron sequences in genes (e.g., self-splicing Group I intron ribozymes) (2–9). The biological Significance BIOPHYSICS AND functions and mechanism of these ribozymes have been discovered COMPUTATIONAL BIOLOGY through structural and biochemical studies. Basedonthe“RNA world” theory, ribozymes likely carried out Currently, the classes of self-cleaving ribozymes consist of Ham- biochemical reactions long before organisms evolved to use merhead, Hairpin, Hepatitis Delta Virus ribozyme (HDV), Varkud protein enzymes as biocatalysts. The continued discovery of new Satellite (VS), glmS ribozyme, Twister, Twister sister, Hatchet, and structures for small self-cleaving ribozymes has shed light on Pistol (10). These classes differ based on their size, structure, and conserved mechanisms in evolution, such as acid–base catalysis cleavage mechanism. Known for their site-specific cleavage, ribo- for self-cleavage reaction. Here, we present the crystal structure zymes with defined biological function include the Hammerhead, of a newly discovered class of self-cleaving ribozymes called VS, and HDV, which all participate in rolling circle replication, Pistol and how it likely uses the phosphoester transfer mecha- whereas the glmS ribozyme functions in controlling gene expression nism for self-cleavage. The results presented here suggest that (11, 12). However, the biological function of a vast majority of the Pistol uses an evolutionarily conserved cleavage mechanism that different self-cleaving ribozymes remains to be explored. is like other self-cleaving ribozymes, such as Twister, Hammer- Through comparative genome analysis, there have been three head, Hairpin, and Hepatitis Delta Virus ribozymes. newly identified classes of self-cleaving ribozymes called Twister sister, Hatchet, and Pistol (3). Biochemical analysis reveals that Author contributions: L.A.N. designed research; L.A.N. performed research; T.A.S. contrib- uted new reagents/analytic tools; L.A.N., J.W., and T.A.S. analyzed data; and L.A.N. and J.W. Pistol can use a variety of divalent metal ions to carry out a com- wrote the paper. plete site-specific, self-cleaving reaction, whereas utilization of The authors declare no conflict of interest. monovalent cations results in modest cleavage rates (3, 10). The − This article is a PNAS Direct Submission. rate of Pistol self-cleavage has been estimated to be >10 min 1 > −1 Data deposition: The crystallography, atomic coordinates, and structure factors have been under physiological conditions and 100 min under optimal deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 5KTJ). magnesium and pH conditions (10). Pistol self-cleavage is via an 1To whom correspondence may be addressed. Email: [email protected] or thomas. internal transesterification reaction, in which the substrate RNA of [email protected]. ′ Pistol G10 2 -hydroxyl on the ribose makes a nucleophillic attack on This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the adjacent 3′-phosphate (Fig. 1B) (3, 10). Self-cleaving ribozymes 1073/pnas.1611191114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1611191114 PNAS | January 31, 2017 | vol. 114 | no. 5 | 1021–1026 Downloaded by guest on September 28, 2021 fold of Pistol necessary to form the active site (Figs. 1A and 2A). In agreement with the biochemistry results, our findings illuminate how two previously described mutations of nucleotides C2 and U3 in the P1 stem of Pistol (Fig. 1B) yielded a ribozyme with reduced cleavage efficiency (10). Based on our structure, we report features of Pistol that were not originally observed in prior secondary structural predictions. One feature is that the P1 and P2 stems are comprised of 5 Watson–- Crick bp instead of 4 bp (Fig. 1B) (10). The P2 stem has an additional U-U wobble base pair between U29 of the enzyme strand and the U11 cleavage site on the substrate strand. The presence of this wobble U-U base pair may help to properly orient the scissile phosphate for the cleavage reaction and could also provide additional stabilization of the U11 base after the cleavage reaction (Fig. 1B). Fig. 1. Overview of the Pistol ribozyme structure. (A) A standard view of Another feature observed in our structure is that Loop 1 con- the Pistol ribozyme is shown in the ribbon diagram. The structural domains tains three highly conserved adenosine nucleobases that form an are colored as follows: P1 is in green, P2 is in blue, P3 is in gray, pseudoknot is A A in magenta, and three loops are in orange. The arrows indicate the 5′ to 3′ A-minor interaction with the P1 stem (Figs. 1 and 4 ). We found direction. (B) A secondary structure of Pistol describes the interaction be- that these three adenosine nucleobases fix the P2 stem in the tween the enzyme strand from nucleotides 1–51 and the substrate strand proper spatial geometry for the active site formation. The P2 stem nucleotides 1–15. The cleavage site of Pistol is between G10 and U11 as in- is after Loop 1 and followed by Loop 2, which flanks the pseu- dicated by the arrow. Our substrate strand contains a noncleavable modified doknot along with Loop 3. Together, Loops 2 and 3 form the active dG base not depicted in this secondary structure. The base numberings are site of Pistol (Fig. 2A), which explains the high sequence conser- in black. vation of nucleotides found in these loops. After the active site, the P3 stem forms 9 Watson–Crick bp and is positioned adjacent to the P2 stem and Loop 3. Finally, the sharp turn in the substrate strand strand with a single-nucleotide mutation at the site of cleavage between the P2 and P3 stems exposes the scissile phosphate on the [from a guanosine to a deoxyguanosine (dG)]. With the loss of its substrate strand (Fig. 1A). 2′-hydroxyl, G10 can no longer serve as a nucleophile for Pistol to carry out its phosphoester transfer reaction. Active Site of Pistol and Catalytic Mechanism.
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