PHOSPHORYLATION OF BACILLUS-SUBTILIS TRANSCRIPTION FACTOR SPOOA STIMULATES TRANSCRIPTION FROM THE SPOIIG PROMOTER BY ENHANCING BINDING TO WEAK OA BOXES

Activation of the spoIIG promoter at the onset of sporulation in Bacil lus subtilis requires the regulatory protein, Spo0A, which binds to tw o sites in the promoter, sites 1 and 2. Phosphorylation of Spo0A is es sential for the initiation of sporulation. Therefore, we examined the role of Spo0A phosphorylation in spoIIG promoter activation. Phosphory lation of Spo0A stimulated transcription from the spoIIG promoter in v itro. In DNAse I footprinting experiments with the spoIIG promoter, we found that phosphorylation of Spo0A increased its affinity for site 2 more than for site 1, which is the site to which nonphosphorylated Sp o0A binds most avidly. This result could not be explained by increased cooperativity between Spo0A bound at sites 1 and 2 because the increa sed affinity for site 2 by phosphorylated Spo0A was also observed with a deletion derivative of the spoIIG promoter containing only site 2. We have located Spo0A-binding sequences in the spoIIG promoter by DMS protection assays and mutational analysis, and found that site 1 conta ins one higher-affinity binding sequence whereas site 2 contains two w eaker-binding sites. Two substitutions in site 2 of the spoIIG promote r that change the sequence to be more like an optimal Spo0A-binding si te were found to increase promoter activity. Moreover, phosphorylation of Spo0A was not required in vivo for activation of the spoIIG promot er containing these strong binding sites. The results suggest that the primary role for phosphorylation of Spo0A is to increase its affinity for specific sites rather than to activate an activity of Spo0A that acts on RNA polymerase at promoters.