Promoter characterization and constitutive expression of the Escherichia coli gcvR gene
Promoter characterization and constitutive expression of the Escherichia coli gcvR gene
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DOI：
10.1128/jb.180.7.1803-1807.1998
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发表时间：
1998-04-01
影响因子：
3.2
通讯作者：
Stauffer, GV
中科院分区：
文献类型：
--
作者：
Ghrist, AC;Stauffer, GV
大肠杆菌甘氨酸裂解抑制蛋白(GcvR)负调控甘氨酸裂解操纵子(GCV)的表达。在本研究中，通过对GcvR-LacZ融合蛋白的N末端氨基酸序列分析，确定了GcvR的翻译起始点。对gcvR启动子区域的引物延伸分析发现，在UUG翻译起始点上游27bp处有一个初级转录起始点，在翻译起始密码子上游约100bp处有一个次要转录起始点。通过突变分析确定初级转录起始点上游的-10和-35启动子区域。GcvR-LacZ融合的表达在甘氨酸或肌苷的存在下没有改变，这两种已知分子分别诱导或抑制GCV的表达。此外，GcvR-LacZ的表达既不受甘氨酸裂解激活蛋白(GCVA)的调节，也不受GcvR自身调节。根据DNA序列分析，推测下游bcp基因的翻译起始密码子与gcvR终止密码子重叠，提示这些基因可能形成操纵子。然而，gcvR-10启动子区域的向下突变对下游bcp-lacZ融合的表达没有影响，对bcp启动子区域的引物延伸分析表明，bcp在gcvR编码序列中有自己的启动子。这些结果表明，gcvR和bcp不形成操纵子。此外，染色体上bcp的缺失对GCV-LacZ的表达没有影响。
The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the gcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the bcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.