FIGURE 6.

Omission of the size-exclusion chromatography step results in sub-stoichiometric contamination of B. subtilis Rel preparations with E. coli ribosomal particles. (A) SDS-PAGE analysis of wild-type full-length Rel protein purified either as described in Figure 5 or with the SEC step omitted (no SEC). Denaturing agarose gel (B) and negative staining electron microscopy (C) analyses of full-length Rel protein purified with and without the SEC step. Individual ribosomal particles are indicated with white arrows on (C). Effects of the SEC omission on synthetase (D) and hydrolase (E) activity of B. subtilis Rel. The synthetase activity was assayed with either Rel alone or in the presence of 0.5 μM 70S, 70S supplemented with 2 μM deacylated tRNA^Val and no mRNA or starved ribosomal complexes (0.5 μM 70S IC(MVF) supplemented with 2 μM deacylated tRNA^Val). The error bars represent standard deviations of the turnover estimates by linear regression using four data points.