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T E Horton

Publications and source records attributed to T E Horton.

5 recordsLinked to original sources
PubMed2000

Cobalt hexammine inhibition of the hammerhead ribozyme.

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.

Binding, Competitive↗
PubMed2000

Metal interactions with a GAAA RNA tetraloop characterized by (31)P NMR and phosphorothioate substitutions.

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.

Magnetic Resonance Spectroscopy↗
PubMed2000

Impact of phosphorothioate substitutions on the thermodynamic stability of an RNA GAAA tetraloop: an unexpected stabilization.

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.

Cadmium↗
PubMed1998

Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity.

Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mM triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n1 = 3.7 +/- 0.4, Kd(1) = 4 +/- 1 microM (type 1) and n2 = 5.2 +/- 0.4, Kd(2) = 460 +/- 130 microM (type 2). The high-affinity type 1 sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme:13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent Kd approximately 10 microM is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n1 = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to > 8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These measurements indicate that, over this NaCl concentration range, hammerhead ribozyme activity is influenced by population of a specific set of divalent cation sites.

Base Sequence↗