Leader region of the gene encoding DNA polymerase III of Bacillus subtilis

Previously we cloned and sequenced the polC gene of Bacillus subtilis and identified regions corresponding to various catalytic domains of DNA polymerase III, the enzyme it encodes. In the present study, by using primer extension, we have identified the transcription start site and a 139 nucleotide leader region upstream of the first codon. This region contains a DnaA box in the non-transcribed DNA strand. An RNA transcript of the leader would contain a sequence that could form a 29 bp stem-loop secondary structure followed by a strong terminator sequence, rich in uracil, before the ribosome binding site. Plasmids were constructed containing either the intact leader region or deletion mutations of the leader, fused to the Escherichia coli lacZ gene in an expression vector. Single copies of the fusions were then integrated into the B. subtilis genome by transformation. Studies of the expression of beta-galactosidase by the transformed cells supported the idea that the leader region is important in regulating polC gene expression.