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. 2007 Mar 13;35(6):2060–2073. doi: 10.1093/nar/gkm005

Table 4.

Cleavage rates for B. subtilis holoenzymes reconstituted in vitro at low Mg2+ concentrations

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.