The P4 metal binding site in RNase P RNA affects active site metal affinity through substrate positioning

Abstract

Although helix P4 in the catalytic domain of the RNase P ribozyme is known to coordinate magnesium ions important for activity, distinguishing between direct and indirect roles in catalysis has been difficult. Here, we provide evidence for an indirect role in catalysis by showing that while the universally conserved bulge of helix P4 is positioned 5 nt downstream of the cleavage site, changes in its structure can still purturb active site metal binding. Because changes in helix P4 also appear to alter its position relative to the pre-tRNA cleavage site, these data suggest that P4 contributes to catalytic metal ion binding through substrate positioning.

FIGURE 1.

Intermolecular photocross-linking analysis of P4 position relative to pre-tRNA. (A) Secondary structures of wild-type and mutant forms of helix P4 used in this study. A gray U indicates the position of the bulged uridine. (B) PAGE analysis of the products resulting from 366-nm irradiation of 0.8 μM pre-tRNA (left), 0.4 μM 4sU69 P RNA (middle), and a mixture of 0.8 μM pre-tRNA and 0.4 μM nM 4sU69 P RNA (right) in 1 M NH₄Cl, 50 mM MES (pH 5.5), 25 mM CaCl₂, 0.02% NP40. The locations of pre-tRNA, P RNA, and the XLU69 intermolecular cross-link are indicated. The position of an intramolecular cross-link is indicated by an asterisk. (C) Analysis of the catalytic activity of the XLU69 cross-link. The gel shows resolution of the gel purified XLU69 RNA (left), the products resulting from incubation of XLU69 in Mg²⁺ (middle), and a 5′-leader sequence marker (right). (D) Alkaline hydrolysis mapping of 5′-end-labeled pre-tRNA in the XLU69 cross-linked RNA. A 5′-leader RNA is shown as a marker. The positions of nucleotides corresponding to N(+1) and N(+4) relative to the RNase P cleavage site are indicated. (E) Analysis of the products resulting from irradiation of a mixture of 0.8 mM pre-tRNA and 0.4 mM 4sU68 P RNA under the conditions noted in B (middle), and analysis of the catalytic activity of purified XLU68 cross-link as described in C (right). A 5′-leader RNA is shown as a marker (left). (F) Alkaline hydrolysis mapping of 5′-end-labeled pre-tRNA in the XLU68 cross-linked RNA. A 5′-leader RNA is shown as a marker. The position of nucleotide corresponding to N(−2) is indicated.

FIGURE 2.

Quantitative phosphorothioate rescue analysis of the effects of mutations in the P RNA catalytic core. (A) Cd²⁺ concentration dependence of k _rel (k _sulfur/k _oxygen) for cleavage of pre-tRNA^ASP with a cleavage site R_P phosporothioate modification by native P RNA (circle), and mutants A248U (square), G332U (triangle), and U69A (diamond) plotted as a function of a nonlinear form of the Hill equation as described previously (Shan et al. 2001). The effect of Cd²⁺ on rate is expressed as the ratio of the observed reaction rate for the phosphorothioate substituted substrate to the corresponding unmodified control (k _rel), which controls for nonspecific effects of Cd²⁺ binding on the reaction. (B) Cd²⁺ concentration dependence of k _rel for cleavage of the Rp phosporothioate-modified substrate by the Δ69 (circle), Δ69U68 (diamond), and Δ69U70 (triangle) mutant RNase P ribozymes. Solid black line reflects a plot of the data from Δ69U68 RNase P RNA plotted with n _Hill fixed at 2. Note that all reactions were carried out in a constant background of 10 mM MgCl₂ to ensure RNA folding and that there was essentially no inhibition of the observed reaction rate due to the presence of Cd²⁺ even at the highest concentrations tested (40 mM).

FIGURE 3.

Three-dimensional representation of Type B RNase P and tRNA adapted from Kazantsev et al. (2005, © National Academy of Sciences, USA). Colored dots in blue, green, orange, and purple represent interactions previously identified between ribozyme and substrate. Yellow dots represent positions involved in the XLU69 cross-link obtained here using E. coli (Type A) RNase P. A black arrow indicates the site of pre-tRNA cleavage.