Biochemical analyses of glmS ribozymes from B. subtilis and B. cereus. (A) Secondary structure model of B. cereus 201 glmS RNA. The G residue marked with an asterisk was introduced to improve the yield of transcription in vitro. (B) Effect of GlcN6P concentration on the rate of self-cleavage of the glmS ribozyme from B. cereus (filled circles). Data corresponding to the 246 glmS ribozyme from B. subtilis are shown for comparison (open circles). (Inset) Effect of Tris-HCl buffer on the rate of self-cleavage in the absence of GlcN6P. 5′ 32P-labeled B. cereus 201 glmS RNA was incubated for 4 h in reactions buffered to pH 7.5 with various concentrations of HEPES or Tris as indicated in mM. NR designates no reaction. Precursor and 5′ cleavage product bands are indicated. (C,D) Effects of MgCl2 concentration (c) and pH on rate constants under otherwise standard assay conditions. Reactions from pH 7.0–9.0 were buffered with Tris, and those below pH 7.0 were buffered with 2-[N-morpholino]ethanesulfonic acid (MES). Data corresponding to B. cereus and B. subtilis glmS ribozymes are represented as in B.
