PDBsum entry 1zft

RNA

PDB id: 1zft

Contents

DNA/RNA

Ligands

SO4

NCO ×2

Waters ×33

References listed in PDB file

Key reference

Title

Water in the active site of an all-Rna hairpin ribozyme and effects of gua8 base variants on the geometry of phosphoryl transfer.

Authors

J.Salter, J.Krucinska, S.Alam, V.Grum-Tokars, J.E.Wedekind.

Ref.

Biochemistry, 2006, 45, 686-700. [DOI no: 10.1021/bi051887k]

PubMed id

16411744

Abstract

The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were determined by X-ray crystallography in a resolution range between 2.3 and 2.7 A. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 A resolution. The native structure revealed a scissile bond angle (tau) of 158 degrees, which is close to the requisite 180 degrees "in-line" geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine), and G8(uridine) folded properly, but exhibited nonideal scissile bond geometries (tau ranging from 118 degrees to 93 degrees) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A-1 sugar pucker. In this regard, variants A8 and U8 appeared to represent nonproductive ground states in which their 2'-OH groups mimicked the pro-R, nonbridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A-1, lending support to the hypothesis that solvent may play an important role in the reaction.

Secondary reference #1

Title

Crystallization and X-Ray diffraction analysis of an all-Rna u39c mutant of the minimal hairpin ribozyme.

Authors

V.Grum-Tokars, M.Milovanovic, J.E.Wedekind.

Ref.

Acta Crystallogr D Biol Crystallogr, 2003, 59, 142-145.

PubMed id

12499551

Secondary reference #2

Title

Functional involvement of g8 in the hairpin ribozyme cleavage mechanism.

Authors

R.Pinard, K.J.Hampel, J.E.Heckman, D.Lambert, P.A.Chan, F.Major, J.M.Burke.

Ref.

EMBO J, 2001, 20, 6434-6442. [DOI no: 10.1093/emboj/20.22.6434]

PubMed id

11707414

Figure 2.
Figure 2 Activity of ribozymes with G8 modifications. Single-turnover cleavage rate constants (single or double exponential) were determined for the two-way junction trans-cleavage construct shown in Figure 1A.

Figure 5.
Figure 5 Reaction mechanisms for phosphodiester bond cleavage involving G8. (A) RNA cleavage catalyzed by the keto -enol tautomerization of G8 is achieved by abstraction of a proton from the 2'-OH by the 6-keto group of G and donation of a proton from the N1 to the 5'-O leaving group. (B) Imino -amino tautomerization cleavage mechanism involving 2,6-diaminopurine at position 8. (C) The imino proton of G8 facilitates nucleophilic attack of the phosphorus by donating a hydrogen bond to a non-bridging phosphate oxygen, indicated by a dashed line, thereby rendering the phosphorus more electropositive. (D) Hydrogen bonding between O6 and 2'-H. The imino proton of G8 and 2'-O polarizes and destabilizes the 2'-O -H bond: base abstraction of the 2'-H by O6 or the action of an unidentified general base results in bond cleavage. (E) General base and general acid catalysis by the N1 of G8.

The above figures are reproduced from the cited reference which is an Open Access publication published by Macmillan Publishers Ltd