ABSTRACT
The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1-2 equiv of Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of =0.6 ppm with addition of up to 10 equiv of Cd(2+). No significant shifts are observed for an RNA.RNA duplex with a single, internal phosphorothioate modification upon addition of Cd(2+). Data obtained using model compounds including diethyl phosphate/thiophosphate, AMP, and AMPS, show that a Cd(2+)-S interaction yields an upfield shift for the (31)P NMR resonance, even in the case of a weak coordination such as with diethyl thiophosphate. Taken together, these data predict that Cd(2+) has a high affinity for the A9 site and suggest that there is flexibility in metal coordination within the binding pocket. Cd(2+) interactions with the cleavage site P1.1-S positions are weaker and appear to be stereospecific. These data have implications for mechanisms that have been proposed to explain the influence of metal ions on hammerhead ribozyme activity. These experiments also show the potential utility of (31)P NMR spectroscopy in conjunction with phosphorothioates as a probe for metal binding sites in nucleic acids.
Subject(s)
Metals/chemistry , Phosphates/chemistry , RNA, Catalytic/chemistry , Thionucleotides/chemistry , Binding Sites , Cadmium/chemistry , Hydrolysis , Magnesium/chemistry , Nuclear Magnetic Resonance, Biomolecular , Organophosphates/chemistry , Phosphorus IsotopesABSTRACT
A metal site in a 5'-GAAA-3' tetraloop, a stabilizing and phylogenetically conserved RNA motif, is explored using (31)P NMR spectroscopy and phosphorothioate modifications. Similar to previous reports [Legault, P., and Pardi, A. (1994) J. Magn. Reson., Ser. B 103, 82-86], the (31)P NMR spectrum of a 12-nucleotide stem-loop sequence 5'-GGCCGAAAGGCC-3' exhibits resolved features from each of the phosphodiester linkages. Titration with Mg(2+) results in distinct shifts of a subset of these (31)P features, which are assigned to phosphodiesters 5' to A6, A7, and G5. Titration with Co(NH(3))(6)(3+) causes only a slight upfield shift in the A6 feature, suggesting that changes caused by Mg(2+) are due to inner-sphere metal-phosphate coordination. R(p)-Phosphorothioate substitutions introduced enzymatically 5' to each of the three A residues of the tetraloop provide well-resolved (31)P NMR features that are observed to shift in the presence of Cd(2+) but not Mg(2+), again consistent with a metal-phosphate site. Analysis of (31)P NMR spectra using the sequence 5'-GGGCGAAAGUCC-3' with single phosphorothioate substitutions in the loop region, separated into R(p) and S(p) diastereomers, provides evidence for an inner-sphere interaction with the phosphate 5' to A7 but outer-sphere or structural effects that cause perturbations 5' to A6. Introduction of an R(p)-phosphorothioate 5' to A7 results in a distinct (31)P NMR spectrum, consistent with thermodynamic studies reported in the accompanying paper that indicate a unique structure caused by this substitution. On the basis of these results and existing structural information, a metal site in the 5'-GAAA-3' tetraloop is modeled using restrained molecular dynamics simulations.
Subject(s)
Metals/chemistry , RNA/chemistry , Magnetic Resonance Spectroscopy , Nucleic Acid Conformation , Phosphorus RadioisotopesABSTRACT
This study analyzes the impact of phosphorothioate substitutions on the thermodynamic stability of a 12-nt RNA hairpin containing a (5')GAAA(3') tetraloop. The thermodynamic consequences of stereospecific phosphorothioate substitutions 5' to each adenosine in the loop region are measured using optical melting and calorimetry experiments. Surprisingly, a single stereospecific phosphorothioate substitution 5' to the second adenosine of the tetraloop, R(p)-A7, results in a stabilization corresponding to a Delta(DeltaG(37)(degrees)(C)) of approximately -2.9 kcal mol(-1) (0.1 M NaCl) when compared with that of an unmodified sample. Five other phosphorothioate-substituted samples did not show significant thermodynamic differences in comparison with the unsubstituted samples. Addition of Mg(2+) to all of the hairpins studied results in increased t(m's) that are fit with a general electrostatic model to a dissociation constant of K(d)(Mg(2+)) approximately 2-3 mM (0.1 M NaCl). The R(p)-A7 phosphorothioate-substituted hairpin showed an unusual decrease in t(m) and apparent increase in enthalpy of unfolding upon addition of Cd(2+). These results may impact the interpretation of interference mapping experiments that use phosphorothioate substitutions to characterize RNAs in solution.