CONTRIBUTIONS OF THE DOMAINS OF THE BACILLUS-SUBTILIS RESPONSE REGULATOR SPO0A TO TRANSCRIPTION STIMULATION OF THE SPOIIG OPERON

Spo0A is a response regulator that controls entry into sporulation by specifically stimulating or repressing transcription of critical devel opmental genes. Response regulators have at least two domains: an outp ut transcription regulation domain and a receiver domain that inhibits the output domain, Phosphorylation of the receiver domain relieves th e inhibition. We examined the in vitro transcription activation mechan ism for Spo0A, phosphorylated Spo0A (Spo0A-P), and a deletion mutant t hat consists solely of the C-terminal output domain (Spo0ABD). Both Sp o0A-P and Spo0ABD stimulated transcription from the spoIIG promoter 10 -fold more efficiently than Spo0A Spo0A-P and Spo0ABD induced DNA dena turation by RNA polymerase in the -10 recognition region, whereas Spo0 A did not. DNase I footprint assays revealed that phosphorylation enha nced binding of intact Spo0A to the OA boxes, while the binding of Spo 0ABD was similar to that of Spo0A Thus, activation of Spo0A by phospho rylation is not primarily due to enhanced DNA binding. The presence of a phosphorylated N terminus increased the stability of the ternary co mplex at the spoIIG promoter. We propose that the primary effect of ph osphorylation is to expose an RNA polymerase interaction domain to pro mote transcription from PspoIIG.