Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes

Abstract

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2′ nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2′-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2′ of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.

Crucial Roles of Two Hydrated Mg2+ Ions in Reaction Catalysis of the Pistol Ribozyme

“…Furthermore,our TS analogue structure strongly suggests the bidentate interaction of G40 with the phosphorane TS during cleavage.I nt he vanadate complex both, N1 of G40 and N2 of G40 are within hydrogen-bonding distance to the 2'O( 2.5 )a nd the pro-R oxygen atom (3.4 ), respectively (Figure 2c,d). Ad irect role of G40 as abase in general acidbase catalysis is widely accepted according to functional mutagenesis assays, [24,31,33] and it is now complemented and consistent with the structural framework provided here.W e also note that the cleavage rate differences observed for phosphorothioate substrates are consistent with our structures. [33,34] Thesignificantly reduced rate measured for the R P diastereomer is in agreement with adirect recognition of the phosphorothioate moiety by G40.…”

“…Ad irect role of G40 as abase in general acidbase catalysis is widely accepted according to functional mutagenesis assays, [24,31,33] and it is now complemented and consistent with the structural framework provided here.W e also note that the cleavage rate differences observed for phosphorothioate substrates are consistent with our structures. [33,34] Thesignificantly reduced rate measured for the R P diastereomer is in agreement with adirect recognition of the phosphorothioate moiety by G40. Furthermore,b ecause the activity was not restored by addition of Mn 2+ ions, [35,36] watermediated (outersphere) rather than innersphere coordination between the phosphorothioate and the metal ion is likely and the M 2+ -'proR-O' distance of 3.3 in our TS structure supports this notion further.…”

“…By further revisiting the pre-cleavage structure with respect to the exact location of this Mg 2+ ion, we noticed that the O6 of G40 is also at ad istance that would allow outershell interactions,a nd hence,m ay contribute to activation of G40 (the general base). Since this hypothesis has not been experimentally challenged in former studies, [31,33] we set out to synthesize aG 40Ap mutant which lacks the O6, but otherwise provides the imine N1 and amine N2 (as encountered in the enol form of G; Figure 6a). Qualitative HPLC cleavage assays indicated that the G40Ap mutant cleaved as fast as the wildtype ribozyme (Figure 6b,c).…”

“…Another nucleotide,w hose role in pistol ribozyme cleavage has been intensively debated, is G42. [18,31,33] In the precatalytic structure,t his guanine mediates the formation of ac left (to accommodate the cleavage site), characterized by the hydrogen bond between the G42 N2 and the 2'Oo f ribose-32 (2.7 ), and by locating its O6 nearly equidistant to the G40 N2 (2.8 )a nd dG53 N2 (2.7 ;s ee Figure S7a). In the TS analogue,G 42 retains the interaction with ribose-32 (2.4 )b ut simultaneously releases G53 and G40 (see Figure S7b).…”

“…The crucial role of the ribose-32 2'OH is consistent with signifi-cantly reduced activities when this group was replaced by either H, OCH 3 ,orN H 2 . [24,31,33,37] Our new structures of TS analogue and product significantly improve the mechanistic understanding of pistol ribozyme phosphodiester cleavage,f or which we postulate the following scenario:Inthe precursor,adivalent ion (Mg 2+ ) becomes innersphere-coordinated to N7 of G33 and is additionally held in place by first shell water-mediated hydrogen-bond interactions,o ne to the 2'Oo fr ibose-32, and the other one to the pro-R oxygen of the scissile phosphate (Figure 2b). At the same time,G 40 assists in proton transfer from the G53 2'O, which attacks the scissile phosphorus atom in-line to the PÀO5' bond.…”