Goss T J, Datta P
Mol Gen Genet. 1985;201(2):308-14. doi: 10.1007/BF00425676.
The biodegradative threonine dehydratase gene (tdc) of Escherichia coli was cloned by isolating a dehydratase negative mutant after Tn5 mutagenesis, cloning the tdc::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoter-like elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P2, was located immediately upstream from the tdc coding region, and a second, P1, was approximately 1 kilobase upstream from P2. Deletion of the potential CAP-binding site from P1 prevented tdc gene expression. However, removal of P2 and a large segment of the upstream DNA had no discernible effect on dehydratase synthesis. A 936-base pair open reading frame was found between P1 and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that P1, and not P2, is necessary for tdc gene expression, and that the DNA sequences coding for the 32 KD polypeptide and threonine dehydratase are part of a single transcriptional unit.
通过在Tn5诱变后分离出一种脱水酶阴性突变体，将tdc::Tn5 DNA克隆到pBR322中，然后在体内替换质粒上的Tn5元件，从而克隆了大肠杆菌的生物降解性苏氨酸脱水酶基因（tdc）。亚克隆和核苷酸序列数据显示出两个不同的原核生物启动子样元件，每个元件都包含一个潜在的CAP结合位点和富含AT的区域，以及一个Shine-Dalgarno序列。这些推定的启动子之一，P2，位于tdc编码区的紧邻上游，另一个，P1，位于P2上游约1千碱基处。从P1中删除潜在的CAP结合位点会阻止tdc基因表达。然而，去除P2和上游DNA的一大段对脱水酶合成没有明显影响。在P1和tdc编码区之间发现了一个936个碱基对的开放阅读框，它产生了一种约32千道尔顿的多肽。数据表明，tdc基因表达需要P1而不是P2，并且编码32 KD多肽和苏氨酸脱水酶的DNA序列是单个转录单元的一部分。
