Identification of a suppressor sequence for DNA replication in the replication origin region of the Bacillus subtilis...

The first replicating fragment of the Bacillus subtilis chromosome, B7, inhibited the replication of the plasmid that carried this fragment. In earlier work using sequential cleavage by Alu I, the suppressor function was located within a 489-base-pair segment. The nucleotide sequence of the entire segment now has been determined. The sequence is characterized by two promoter-like structures and several putative recognition sequences, such as termination signals, 2-fold symmetries, inverted repeats, and repeats. By means of sequential cleavage with exonuclease BAL-31, the essential region for suppression was located in a 200-base-pair region that contains the two promoters with the same orientation. Specific transcription was produced in vitro by using B. subtilis or Escherichia coli RNA polymerases. The transcription was mostly from the second promoter. Elimination of the -35 region of the second promoter dramatically affected both inhibitory activity and in vitro transcription, suggesting that the transcriptional activity of the second promoter is involved in the cis-inhibition of DNA replication. The significance of the suppressor sequence in the region of the replication origin of the B. subtilis chromosome is discussed.