Comparative study on tertiary contacts and folding of RNase P RNAs from a psychrophilic, a mesophilic/radiation-resistant, and a thermophilic bacterium

Multiple turnover kinetics of pre-tRNA^Gly (200 nM) cleavage by T. thermophilus P RNA (20 nM) at 20 mM Mg(OAc)₂, 100 mM NH₄OAc, 50 mM Hepes, pH 7.0, were conducted at 37°C, but using different preincubation procedures. Curve a: preincubation of P RNA for 15 min at 37°C before substrate addition. Curve b: preincubation of P RNA for 15 min at 55°C before substrate addition. Curve c: preincubation of P RNA for 5 h at 37°C before substrate addition. Curve d: preincubation of P RNA for 15 min at 37°C; after addition of substrate, the reaction was further preincubated for 285 min (4.75 h; without withdrawal of aliquots), whereupon fresh substrate (200 nM) was added (final pre-tRNA plus mature tRNA concentration: 400 nM) and the processing reaction monitored for another period of 285 min.