Table 3.
Cleavage activity of B. subtilis wt and mutant RNase P holoenzymes acting on ptRNAGly wt and G74 or G75 mutant substrates at 2 mM Mg2+ and two protein concentrations
| P RNA | ptRNAGly | B. subtilis P protein [nM] | kobs | krel |
|---|---|---|---|---|
| B. subtilis wt | wt | 37.5 | 0.18 ± 0.03 | 1.0 |
| B. subtilis wt | wt | 62.5 | 0.20 ± 0.04 | 1.1 |
| B. subtilis C258 | wt | 37.5 | (4.0 ± 2.0) × 10−4 | 2.2 × 10−3 |
| B. subtilis C258 | wt | 62.5 | (1.3 ± 0.5) × 10−3 | 7.2 × 10−3 |
| B. subtilis C259 | wt | 37.5 | (5.0 ± 1.0) × 10−4 | 2.8 × 10−3 |
| B. subtilis C259 | wt | 62.5 | (1.6 ± 0.9) × 10−3 | 8.9 × 10−3 |
| B. subtilis wt | G74 | 37.5 | (7.0 ± 1.0) × 10−4 | 3.9 × 10−3 |
| B. subtilis wt | G74 | 62.5 | (1.3 ± 0.3) × 10−3 | 7.2 × 10−3 |
| B. subtilis C258 | G74 | 37.5 | (2.0 ± 1.0) × 10−4 | 1.1 × 10−3 |
| B. subtilis C258 | G74 | 62.5 | (3.0 ± 2.0) × 10−4 | 1.7 × 10−3 |
| B. subtilis C259 | G74 | 37.5 | 0.15 ± 0.006 | 0.8 |
| B. subtilis C259 | G74 | 62.5 | 0.30 ± 0.04 | 1.7 |
| B. subtilis wt | G75 | 37.5 | (3.0 ± 1.0) × 10−4 | 7.2 × 10−3 |
| B. subtilis wt | G75 | 62.5 | (4.0 ± 3.0) × 10−4 | 2.2 × 10−3 |
| B. subtilis C258 | G75 | 37.5 | (6.0 ± 4.0) × 10−4 | 3.3 × 10−3 |
| B. subtilis C258 | G75 | 62.5 | (1.8 ± 0.2) × 10−3 | 1.0 × 10−2 |
| B. subtilis C259 | G75 | 37.5 | (1.4 ± 0.8) × 10−4 | 0.6 × 10−3 |
| B. subtilis C259 | G75 | 62.5 | (5.0 ± 0.1) × 10−4 | 2.8 × 10−3 |
Assay conditions: 50 mM Mes pH 6.1, 100 mM KCl, 2 mM MgCl2; P RNA concentration was 20 nM, protein concentration as indicated and substrate concentration was 200 nM; 5′-endlabeled substrate was added in trace amounts (<1 nM). kobs is given in pmol substrate converted per pmol of P RNA per min; for krel, individual kobs values were divided by kobs for the wt P RNA at 37.5 nM P protein and acting on ptRNAwt (first line); values are based on at least three independent experiments.