Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme

Abstract

Reaction-intermediate analogs have been used to understand how phosphoryl-transfer enzymes promote catalysis. Herein we report the first structure of a small ribozyme crystallized with a 3′-OH, 2′,5′-linkage in lieu of the normal phosphodiester substrate. The new structure shares features of the reaction coordinate exhibited in prior ribozyme structures including a vanadate complex that mimicked the oxyphosphorane transition state. As such, the structure exhibits reaction-intermediate traits that allow direct comparison of stabilizing interactions to the 3′-OH, 2′,5′-linkage contributed by the RNA enzyme and its protein counterpart, ribonuclease. Clear similarities are observed between the respective structures including hydrogen bonds to the non-bridging oxygens of the scissilephosphate. Other commonalities include carefully poised water molecules that may alleviate charge build-up in the transition state and placement of a positive charge near the leaving group. The advantages of 2′,5′-linkages to investigate phosphoryl-transfer reactions are discussed, and argue for their expanded use in structural studies.

Structural effects of nucleobase variations at key active site residue Ade38 in the hairpin ribozyme

“…5A,B). Thus, the Cyt38 variants further corroborated the hairpin ribozyme propensity to position a hydrogen-bond donating group in the pocket occupied by N6 of Ade38, as observed in both the WT reaction-intermediate (29,59) and transition-state (oxo-vanadium) mimic structures (Torelli et al 2007(Torelli et al , 2008. In this manner, the N4 amine of Cyt38 was poised to engage in a 3.4 Å hydrogen-bond interaction with the pro-Rp oxygen (equivalent) of Gua+1 in the 29,59 structure ( Fig.…”

“…3G, 7A). This observation demonstrates the extreme favorability of accommodating a negatively charged atom in this pocket, which is filled by the pro-Rp oxygen equivalent of the scissile bond in reaction-intermediate and transition-state-analog complexes (Torelli et al 2007(Torelli et al , 2008. Interestingly, nucleobase variants AP38 and Gua38, which are devoid of an N6 exocylic amine equivalent, did not exhibit water molecules at site 52.…”

“…Experimental and computational evidence continues to suggest a crucial role for water in the mechanism of action of the hairpin ribozyme (Pinard et al 2001;Kuzmin et al 2005;Rhodes et al 2006;Salter et al 2006;Torelli et al 2007Torelli et al , 2008. A summary of active site waters observed in the respective precatalytic and reaction-intermediate constructs of this investigation has been assembled for comparison to the previously reported WT coordinates (Fig.…”

“…This work was conducted in the context of a minimal, hinged hairpin ribozyme construct reported previously (MacElrevey et al 2007;Torelli et al 2007Torelli et al , 2008Fig. 2).…”

“…The trend in reactivity did not appear to be the result of changes from an N1 to N3 moiety or to be correlated strictly with the substituted group's imino pK a . Moreover, no effort was made to relate the substituted nucleobase to its propensity to favor the syn conformation, which is preferred by Ade38 in the native state (Rupert and Ferré-D'Amaré 2001;Rupert et al 2002;Alam et al 2005;Salter et al 2006;MacElrevey et al 2007;Torelli et al 2007Torelli et al , 2008. Although it is tempting to accept such modifications as pure indicators of chemical functionality, previous studies indicated that single nucleobase changes could evoke large, unforeseen conformational alterations, thus obfuscating the direct assignment of catalytic determinants (Alam et al 2005;Salter et al 2006).…”