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 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.
Rowe-Magnus
D A
DA
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Spiegelman
G B
GB
D001412:Bacillus subtilis / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006493:Heparin / Q000494:pharmacology; D009691:Nucleic Acid Denaturation / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics*; D013171:Spores, Bacterial / Q000235:genetics; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012808:Sigma Factor; C080950:Spo0A protein, Bacillus subtilis; D014157:Transcription Factors; C063202:sporulation-specific sigma factors; D006493:Heparin; D012321:DNA-Directed RNA Polymerases