Conformational Heterogeneity at Position U37 of an All-RNA Hairpin Ribozyme with Implications for Metal Binding and the Catalytic Structure of the S-Turn^,

Abstract

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

“…recall G8I/2'-OMe A−1 and G8I/2'-dA−1 revealed little change). Overall, subtle changes were observed in the conformation of the A38 base, which mirrored the results of a prior study in which U39 was replaced by a propyl linker (11). A comparison of the N1 positions of A38 from the most active conformation (i.e.…”

“…The closest potential Hbond partner is the O4' oxygen of residue A9, but this group is ∼3.6 Å away in the native and variant structures (on average). At present, it is unclear why the native G8 structure does not corroborate the solution data, although a previous structural study of the hairpin ribozyme supported the possibility that the S-turn motif in the crystal structure represented a folding intermediate (11). The G8I substitution itself was reported previously to cause a +2.2 kcal/mol increase in ΔΔG of the hairpin ribozyme during catalysis, as well as a 'strong' effect by NAIM (45,46).…”

“…The 4WJ form of the hairpin ribozyme has been solved in complex with vanadate, thereby providing a spatial analogue for the trigonal bipyramidal transition state of this and other ribozyme reactions (10,23,39). A comparison of the vanadate complex to the native G8 , pre-catalytic structure of this and prior studies (9)(10)(11), revealed that τ = 144° for the vanadate mimic ( Figure 6B & Table 2). Although this angle is not ideal for phosphoryl transfer, several features suggest it is the closest available structure to the actual geometry of the reaction intermediate.…”

“…The minute size of this 61-mer ( Figure 1A) enabled the facile incorporation of single-atom point alterations into the sequence by use of solid support chemical synthesis. This approach led to the identification of conformational heterogeneity at position U37 that could be eliminated by incorporation of a U39C point mutant (11). Overall, the structure of the minimal all-RNA variant ( Figure 1B) exhibited a modest 1.3 Å r.m.s.d.…”

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

“…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).…”