Characterization of RNA-based and protein-only RNases P from bacteria encoding both enzyme types

Comparison of processing activities of P RNAs from T. indicus (thermophile), E. coli (mesophile), and T. maritima (thermophile) at elevated assay temperatures. (A) RNA-alone single turnover (sto) reactions containing 25 nM P RNA and <1 nM 5′-³²P-endlabeled pre-tRNA^Gly were performed in KN buffer supplemented with 100 mM MgCl₂ at 25°C, 50°C, 60°C, or 70°C. Before reaction start, pre-tRNA and P RNA were preincubated separately for 5 min at 55°C and 5 min at the respective assay temperature. (B) Corresponding RNase P holoenzyme reactions; holoenzymes were reconstituted with the RnpA protein from T. maritima. In the holoenzyme reaction, 25 nM P RNA were assembled with 25 nM T. maritima P protein and analyzed in KN buffer supplemented with 4.5 mM MgCl₂ in the presence of 125 nM pre-tRNA substrate (multiple turnover, mto) at 25°C, 50°C, 60°C, or 70°C. The data shown in panels A and B are based on three independent experiments (±SD) each. For RNA preincubation and holoenzyme assembly, see Materials and Methods.