Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

Abstract

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca(2+), Mg(2+), Mn(2+), and Sr(2+) have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.

FIGURE 1.

(A) The wild-type Schistosoma hammerhead construct used for the kinetics studies. (Black) The ribozyme strand; (gray) the substrate strand. The cleavable substrate strand has either a ³²P on the 5′-end or a fluorescein label on the 3′-end and is cleaved at C₁₇, as indicated by the arrow. (B) The noncleavable wild-type Schistosoma hammerhead ribozyme construct used for FRET studies. Cy3 was coupled to a modified U in loop II. Cy5 was coupled to the modified 3′-end of the substrate strand. The substrate strand has a 2′-deoxyribose on C₁₇ to prevent cleavage. (C) The UUCG control construct used for FRET studies. Cy3 was coupled to a modified U in loop II. The same noncleavable substrate strand was used for the UUCG control and the wild-type complex shown in B.

FIGURE 2.

(A) FRET efficiency (E_FRET) versus divalent metal ion concentration for folding of the noncleavable Schistosoma HHRz (filled symbols) wild-type and (open symbols) UUCG control constructs in (circles) Mn²⁺, (diamonds) Mg²⁺, (squares) Ca²⁺, or (triangles) Sr²⁺. (B) FRET efficiency versus monovalent metal ion concentration for folding of the noncleavable wild-type construct in (squares) Na⁺ or (circles) NH₄ ⁺. Error bars for replicate measurements are within the symbols.

FIGURE 3.

(A) Plot of FRET efficiency of the noncleavable wild-type Schistosoma HHRz FRET construct as a function of time in 1 mM Mg²⁺. (B) Plot of fraction cleaved substrate as a function of time for the wild-type Schistosoma HHRz (chemically synthesized Cy3-labeled ribozyme strand and transcribed substrate strand) in 1 mM Mg²⁺. The line represents the fit to Equation 1 and gives a k _obs of 0.003 min⁻¹.

FIGURE 4.

(A) Plot of the pK _a of the hydrated metal ion as a function of the log(k _obs) for the Schistosoma HHRz in 1 mM metal ion, 50 mM Tris, 0.1 M NaCl (pH 7.0). The line represents the best fit between log(k _obs) and pK _a for the Schistosoma HHRz (slope=−0.96, R = 0.93). (B) Plot of the ionic radius of the metal ion as a function of log(k _obs) for the Schistosoma HHRz. (C) Plot of the absolute hardness of the metal ion as a function of log(k _obs) for the Schistosoma HHRz. The values for pK _a, ionic radius, and absolute hardness are from Feig and Uhlenbeck (1999).