RNA is the preferred phosphate acceptor for PhoClp1. Reaction mixtures (10 μL) containing 50 mM Tris-acetate (pH 6.5), 10 mM MgCl2, 500 μM [γ32P]ATP, 5 μM each of 24-mer 5′-OH DNA and RNA oligonucleotides (of equivalent sequences depicted in B), and PhoClp1 as specified in B were incubated for 15 min at 55°C. Control reactions contained 10 U of T4 polynucleotide kinase (Pnk) plus either the DNA or RNA substrate. The products were analyzed by denaturing PAGE and visualized by autoradiography (A). The extents of DNA and RNA phosphorylation were quantified by scanning the gel and are plotted as a function of input PhoClp1 in B. Each datum is the average of three separate titration experiments. Error bars denote the standard deviation.
