The role of a 20 bp conserved region located 45-64 nucleotides 5′ of the transcription start point in and was investigated by deletion analysis and by mobility shift assay. Deletions 5′ of this conserved sequence had little effect on expression of a fusion, whereas deletions extending into the sequence reduced expression of the fusion by 85%. The timing of expression of was not affected by deletion of the sequence. The region was shown by mobility shift assays to bind specifically to a protein. Binding activity was detected in protein extracts prepared from bacteria 1 h or more after they had started to sporulate, but not in extracts prepared from vegetative bacteria. Mutations in all known loci were screened but none prevented appearance of the binding activity; nor did mutations in any of the stage II and III loci tested. It is concluded that the 20 bp conserved region is the binding site of an activator that is subject to temporal regulation independent of known loci.


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