FIGURE 2.

Substrates used for functional assays. (A) Substrates containing a 5′-fluorescein label include pre-tRNA^Asp from Bacillus subtilis and Arabidopsis thaliana pre-tRNA^Cys and pre-tRNA^Gly from the mitochondrial and nuclear genomes, respectively. Structural features of pre-tRNA are detailed on pre-tRNA^Cys, including the 5′ leader, 3′ trailer, and the D- and TψC-loops. Black arrows indicate canonical RNase P cleavage site. (B) Fluorescence anisotropy binding curves for AtPRORP1 binding to B. subtilis pre-tRNA^Asp (log scale). A hyperbola (Equation 1, Materials and Methods) was fit to the data. Data were measured in 30 mM MOPS pH 7.8, 1 mM TCEP, and 20 mM CaCl₂ with 250 mM (▾), 330 mM (▪), 450 mM (♦), and 550 mM NaCl (•). (C) Na⁺ dependence of AtPRORP affinity. Equation 4 (Materials and Methods) was fit to the data. The slope of the line (Z) reports on the apparent number of ionic interactions with substrate phosphodiester bonds, while the intercept [log(K₀)] reports on the nonionic contributions to affinity. Data include AtPRORP1 binding to pre-tRNA^Asp (♦) and pre-tRNA^Cys (•) in 20 mM Ca²⁺, as well as AtPRORP2 in 6 mM Ca²⁺ binding to pre-tRNA^Asp (⋄) and pre-tRNA^Gly (□).