FIGURE 2.

Cleavage analysis in vitro of yeast HRA1 RNA with various RNase P enzymes. (A) Yeast HRA1 RNA was transcribed in vitro after linearization of plasmid DNA by NdeI. Both internal labeled (B) and 5′-end-labeled (C) HRA1 RNA were prepared according to Materials and Methods, and then used for cleavage assays in vitro by catalytic E. coli M1 RNA (lane 2, plus 90 mM MgCl₂), E. coli RNase P holoenzyme (lane 3), and yeast RNase P holoenzyme (lane 4) in PA buffer (50 mM Tris·HCl at pH 7.5, 10 mM MgCl₂, 100 mM NH₄Cl). Labeled HRA1 RNA in buffer PA was used as a negative control (lane 1). Reactions were performed at 37°C for 60 min and stopped by adding 8 M urea dye. Samples were then electrophoresed in a 5% polyacrylamide/8 M urea gel and exposed to Kodak BioMax film.