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1.
FIGURE 6.

FIGURE 6. From: Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.

P protein increases the affinity of metal ions in the active site of P RNA. Interpretation of the Cd2+ rescue data supports a model in which two Mg2+ ions bind to the active site of the enzyme–substrate complex in the ground state. The apparent affinities of the rescuing ion interactions are indicated. However, the affinities of corresponding active site Mg2+ ions may be significantly different. However, as indicated in the text, evaluation of the Mg2+ titration data are consistent with cooperative binding of a high affinity class of metal ions that contributes to catalysis as indicated in the diagram.

Lei Sun, et al. RNA. 2007 September;13(9):1505-1515.
2.
FIGURE 4.

FIGURE 4. From: Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.

Quantitative analysis of Cd2+ dependent rescue of the (+1 Rp) PS substrate modification. Single turnover rate constants were determined at a series of increasing Cd2+ concentrations in the background of 17.5 mM Mg2+. (A) Plots of k obs (RNase P) versus Cd2+ concentration are shown for PS modified (○) and unmodified (•) substrates. (B) Plots of k obs (P RNA alone) versus Cd2+ concentration are shown for modified (○) and unmodified (•)substrates. (C) The k obs (P RNA alone) data for PS modified substrates are shown separately for a better view of Cd2+ stimulation at low Cd2+ concentrations. Data represent the average values for at least three independent trials.

Lei Sun, et al. RNA. 2007 September;13(9):1505-1515.
3.
FIGURE 3.

FIGURE 3. From: Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.

Cd2+ rescues the cleavage of a N(+1) PS substrate modification by P RNA and RNase P holoenzyme. Single turnover reactions were performed under standard conditions (50 mM MES at pH 5.75, 100 mM NaCl, 17.5 mM MgCl2, and 0.005% Triton X-100) in the absence (panels 1,3) or presence of 5 mM Cd2+ (panels 2,4) for RNase P holoenzyme (panels 3,4) and P RNA (panels 1,2) reactions. Aliquots are removed at predetermined time points and analyzed in 20% PAGE. The positions of products resulting from correct cleavage (−1/+1) and miscleavage (−2/−1) are indicated. The product marked by an asterisk results from correct cleavage of a minor population of substrate containing an additional nucleotide at its 5′ end.

Lei Sun, et al. RNA. 2007 September;13(9):1505-1515.
4.
FIGURE 2.

FIGURE 2. From: Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.

Kinetic analyses of the pH and Mg2+ dependence of the single turnover rate constants for P RNA and RNase P holoenzyme cleavage of pre-tRNAMet608. (A) Single turnover reactions for RNase P holoenzyme (•) and P RNA alone (○) are compared at saturating enzyme concentration in: MES 50 mM (pH 5.75), NaCl 100 mM, MgCl2 17.5 mM, and Triton 0.005%. (B) Plots of log(k obs) versus pH for RNase P (•) and P RNA (○) are fit to a linear equation (slope=1.1 and 1.2 for RNase P and P RNA alone reactions, respectively). (C) Plot of k obs values for P RNA and RNase P holoenzyme versus Mg2+ concentration. The data are fit to the equation for a cooperative binding mechanism (Equation [2], Materials and Methods). The Hill coefficients are unity for both reactions; however, the apparent dissociation constant for holoenzyme is smaller than that for P RNA alone (K Mg [RNase P]=32 mM and K Mg [P RNA alone] >250 mM). The inset shows a closeup of the data at low Mg2+ concentrations.

Lei Sun, et al. RNA. 2007 September;13(9):1505-1515.
5.
FIGURE 5.

FIGURE 5. From: Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.

Comparison of k rel as a function of Cd2+ concentration for P RNA and RNase P holoenzyme shows that C5 enhances the binding affinity of catalytic metal ion. (A) Relative reaction rates (k rel) calculated from the ratio of observed reaction rates for substrates with and without the sulfur substitution (i.e., k rel=k S obs/k O obs) are plotted versus Cd2+ concentrations. Data for the holoenzyme reaction is represented by (•) and P RNA alone is represented by (○). Fitting to the Hill equation reveals that n=2 for the holoenzyme reaction, and K Cd (holo)=9 mM and K Cd (P RNA alone)>30mM. (B) Interpretation of the concentration dependent rescue of the Rp phosphorothioate modification by Cd2+. Because the k rel analysis controls for nonspecific effects of Cd2+, the cooperative nature of the Cd2+-dependent rescue suggests that two rescuing metal ions interact with the single sulfur modification, which reflects two metal ions binding in the P RNA active site for the native, unmodified substrate.

Lei Sun, et al. RNA. 2007 September;13(9):1505-1515.
6.
FIGURE 1.

FIGURE 1. From: Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity.

(A) Secondary structure diagram of E. coli P RNA. Helices are designated as P (paired) and numbered from the 5′ end of the RNA. Segments of the RNA connecting helices are designated J (joining) and numbered according to the helices they connect. The helices P1–P18 that make up the secondary structure of E. coli P RNA are labeled. The secondary structure is organized according to the three-dimensional structure of the P RNA subunit, and arrows depict connections that define the path of the RNA chain. (B) Cartoon diagram of the three-dimensional structure of a Type A P RNA with pre-tRNA bound. The structure diagram is based on the crystal structure of Thermotoga maritime P RNA (Torres-Larios et al. 2005). Individual helices are shown as cylinders that are colored according to the secondary structure diagram in panel A. The bound pre-tRNA is depicted by a black ribbon and the P protein subunit is shown as a sphere. The 5′-leader sequence of pre-tRNA is shown as a dashed line. (C) Sequence and secondary structure of pre-tRNAMet608. The location of the RNase P cleavage site between nucleotides N(+1) and N(−1) is indicated by an arrow. (D) Detail of the proposed active site metal ion interactions and the reactive phosphate of pre-tRNA in the transition state. As described in the text, two metal ions are proposed, both of which interact with the pro-Rp phosphate oxygen of the reactive phosphate. This position is substituted by a sulfur atom in the Rp(+1) pre-tRNA substrate.

Lei Sun, et al. RNA. 2007 September;13(9):1505-1515.

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