Gase K, Ellinger T, Malke H
Institute for Molecular Biology, Jena University, Germany.
Mol Gen Genet. 1995 Jun 25;247(6):749-58. doi: 10.1007/BF00290407.
On the Streptococcus equisimilis H46A chromosome, the divergent coding sequences of the genes for the plasminogen activator streptokinase (skc) 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 tracts. To begin to understand the expression control of these two genes, we mapped their transcriptional initiation sites by S1 nuclease analysis and studied the influence of the bent intergenic region on promoter strength, using promoter-reporter gene fusions of skc' and lrp' to 'lacZ from Escherichia coli. The major transcriptional start sites, in both S. equisimilis and E. coli, mapped 22 bases upstream of the ATG start site of lrp (G), and 24 and 32 bases upstream of the translational initiation codon of skc (A and G, respectively), indicating the existence 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. equisimilis dexB gene, thus allowing promoter strength to be measured in multiple plasmid-form copies in the heterologous host and in single-copy genomic form following integration into the skc region of the homologous host. In S. equisimilis, skc'-'lacZ was expressed about 200-fold more 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 lrp 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-fold the activity of the lrp promoter. This comparative promoter analysis shows that skc has a complex promoter structure, the activity of which in the homologous genomic environment specifically depends on sequences upstream of the two core promoters. Thus, the skc promoter structure resembles that of an array of promoters involved in a transcriptional switch; however, the nature of the potential switch factor(s) remains unknown.
在类马链球菌H46A染色体上，纤溶酶原激活剂链激酶（skc）基因和一种富含亮氨酸蛋白（lrp）基因的反向编码序列被一个富含AT序列的328 bp内在弯曲DNA区域隔开，lrp的功能尚不清楚。为了初步了解这两个基因的表达调控，我们通过S1核酸酶分析确定了它们的转录起始位点，并利用skc'和lrp'与大肠杆菌' lacZ的启动子 - 报告基因融合，研究了弯曲基因间隔区对启动子强度的影响。在类马链球菌和大肠杆菌中，主要转录起始位点分别位于lrp（G）的ATG起始位点上游22个碱基处，以及skc的翻译起始密码子（分别为A和G）上游24和32个碱基处，这表明在螺旋的相对面上存在两个串联排列的重叠典型skc启动子。报告基因融合体在大肠杆菌中被克隆到一个含有类马链球菌dexB基因1.1 kb片段的载体上，这样就可以在异源宿主中以多质粒形式拷贝以及整合到同源宿主的skc区域后以单拷贝基因组形式来测量启动子强度。在类马链球菌中，skc'-'lacZ的表达强度比相应的lrp'-'lacZ融合体强约200倍。相比之下，在大肠杆菌中，相应的表达水平仅相差约11倍。删除skc和lrp核心启动子上游的202 bp弯曲区域，导致类马链球菌中skc启动子活性下降13倍，但在该宿主中并未改变lrp启动子强度。相反，在大肠杆菌中研究时，这种缺失并未改变skc双启动子的强度，甚至使lrp启动子的活性提高了2.4至3倍。这种比较启动子分析表明，skc具有复杂的启动子结构，其在同源基因组环境中的活性特别依赖于两个核心启动子上游的序列。因此，skc启动子结构类似于参与转录开关的一系列启动子；然而，潜在开关因子的性质仍然未知。
