Table 4.
P RNA | ptRNAGly | Enzyme concentration | [Mg2+] | kobs | krel |
---|---|---|---|---|---|
B. subtilis wt | wt | 10 nM P RNA/ 37 nM RnpA | 4.5 | 9.7 ± 0.08 | 1.0 |
B. subtilis C258 | wt | 10 nM P RNA/ 37 nM RnpA | 4.5 | 4.4 ± 0.08 | 0.45 |
B. subtilis C259 | wt | 10 nM P RNA/ 37 nM RnpA | 4.5 | 8.4 ± 0.12 | 0.87 |
B. subtilis wt | wt | 10 nM P RNA/ 37 nM RnpA | 2.0 | 4.6 ± 0.09 | 1.0 |
B. subtilis C258 | wt | 10 nM P RNA/ 37 nM RnpA | 2.0 | 0.8 ± 0.01 | 0.17 |
B. subtilis C259 | wt | 10 nM P RNA/ 37 nM RnpA | 2.0 | 1.4 ± 0.03 | 0.30 |
B. subtilis C259 | G74 | 10 nM P RNA/ 37 nM RnpA | 2.0 | 6.5 ± 0.08 | |
B. subtilis wt | wt | 50 nM P RNA/ 185 nM RnpA | 2.0 | 47.7 ± 0.68 | 1.0 |
B. subtilis C258 | wt | 50 nM P RNA/ 185 nM RnpA | 2.0 | 5.9 ± 0.07 | 0.12 |
B. subtilis C259 | wt | 50 nM P RNA/ 185 nM RnpA | 2.0 | 6.2 ± 0.05 | 0.13 |
Assay conditions: 20 mM Hepes pH 7.4 (37°C), 2 mM Mg(OAc)2, 150 mM NH4OAc, 2 mM spermidine, 0.05 mM spermine, 4 mM β-mercaptoethanol, and P RNA and B. subtilis P protein (RnpA) concentrations as indicated; the substrate concentration was 100 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; krel is defined as the ratio of kobs obtained with the mutant versus wt holoenzyme under the respective conditions; values are based on at least four independent experiments.