Abstract
Spo0A is a response regulator that controls entry into sporulation by specifically stimulating or repressing transcription of critical developmental genes. Response regulators have at least two domains: an output transcription regulation domain and a receiver domain that inhibits the output domain. Phosphorylation of the receiver domain relieves the inhibition. We examined the in vitro transcription activation mechanism for Spo0A, phosphorylated Spo0A (Spo0A approximately P), and a deletion mutant that consists solely of the C-terminal output domain (Spo0ABD). Both Spo0A approximately P and Spo0ABD stimulated transcription from the spoIIG promoter 10-fold more efficiently than Spo0A. Spo0A approximately P and Spo0ABD induced DNA denaturation by RNA polymerase in the -10 recognition region, whereas Spo0A did not. DNase I footprint assays revealed that phosphorylation enhanced binding of intact Spo0A to the 0A boxes, while the binding of Spo0ABD was similar to that of Spo0A. Thus, activation of Spo0A by phosphorylation is not primarily due to enhanced DNA binding. The presence of a phosphorylated N terminus increased the stability of the ternary complex at the spoIIG promoter. We propose that the primary effect of phosphorylation is to expose an RNA polymerase interaction domain to promote transcription from PspoIIG.
Highlights
Both Spo0AϳP and Spo0ABD stimulated transcription from the spoIIG promoter 10-fold more efficiently than Spo0A
We demonstrate that Spo0AϳP stimulated a step subsequent to RNA polymerase binding and activation of PspoIIG primarily involved the modification of RNA polymerase-promoter complexes, in the Ϫ10 region of the promoter
The three forms of Spo0A were incubated with RNA polymerase and template to form initiation complexes, and a single round of transcription was permitted using heparin to inhibit further initiation
Summary
Template DNA—The spoIIG template was isolated from the plasmid pUCIIGtrpA [17] on a 600-bp DNA fragment generated by digestion with PvuII. This fragment contained the spoIIG promoter, both upstream Spo0A binding sites, and the trpA transcription terminator approximately 160-bp downstream from the transcription start site. The trpA terminator functioned effectively in vitro. The fragment was isolated by electrophoresis, recovered by electroelution, and stored in 10
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