COMPLEX TRANSCRIPTIONAL CONTROL OF THE STREPTOKINASE GENE OF STREPTOCOCCUS-EQUISIMILIS H46A

On the Streptococcus equisimilis H46A chromosome, the divergent coding sequences of the genes for the plasminogen activator streptokinase (s kc) and a leucine-rich protein (lrp), the function of which is unknown , are separated by a 328 bp intrinsically bent DNA region rich in AT t racts. To begin to understand the expression control of these two gene s, we mapped their transcriptional initiation sites by S1 nuclease ana lysis and studied the influence of the bent intergenic region on promo ter strength, using promoter-reporter gene fusions of skc' and Erp' to 'lacZ from Escherichia coli. The major transcriptional start sites, i n both S. eqzlisimilis and E. coli, mapped 22 bases upstream of the AT G start site of lrp (G), and 24 and 32 bases upstream of the translati onal initiation codon of skc (A and G, respectively), indicating the e xistence of two overlapping canonical skc promoters arranged in tandem on opposite faces of the helix. The reporter gene fusions were cloned in E. coli on a vector containing a 1.1 kb fragment of the S. equisim ilis dexB gene, thus allowing promoter strength to be measured in mult iple plasmid-form copies in the heterologous host and in single-copy g enomic form following integration into the skc region of the homologou s host. In S. equisimilis, skc'-'lacZ was expressed about 200-fold mor e strongly than the corresponding lrp'-'lacZ fusion. In contrast, in E . coli, the corresponding levels of expression differed by only about 11-fold. Deletion of the 202 bp bent region upstream of the skc and lr p core promoters caused a 13-fold decrease in skc promoter activity in S. equisimilis but did not alter lrp promoter strength in this host. In contrast, when studied in E. coli, this deletion did not alter the strength of the skc double promoter and even increased by 2.4- to 3-fo ld the activity of the lrp promoter. This comparative promoter analysi s shows that skc has a complex promoter structure, the activity of whi ch in the homologous genomic environment specifically depends on seque nces upstream of the two core promoters. Thus, the skc promoter struct ure resembles that of an array of promoters involved in a transcriptio nal switch; however, the nature of the potential switch factor(s) rema ins unknown.