Peakman T, Busby S, Cole J
School of Biochemistry, University of Birmingham, England.
Eur J Biochem. 1990 Jul 31;191(2):325-31. doi: 10.1111/j.1432-1033.1990.tb19126.x.
The 74-min region of the Escherichia coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read-through from the Fnr-dependent nirB promoter. No promoter activity was associated with DNA fragments between the BamHI site located within the N-terminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.
大肠杆菌染色体的74分钟区域包含五个已知序列的开放阅读框。这些基因中的第一个和最后一个,即nirB和cysG,转录方向相同,且两者对于依赖NADH的亚硝酸还原酶活性都是必需的。位于nirB和cysG之间的其他基因nirD、nirE和nirC的功能尚不清楚。nirB基因从一个厌氧诱导的启动子转录,其表达依赖于转录激活蛋白Fnr。在此我们表明,nirD、nirE、nirC和cysG基因也从nirB启动子表达。亚克隆cysG后,在cysG上游不到100个碱基处定位到了第二个启动子。通过S1作图在该区域检测到两组间隔40个碱基的转录起始点。在有氧生长以及存在或不存在亚硝酸盐的厌氧生长过程中,分离的cysG启动子的转录速率相同。然而,当nirB基因及其启动子克隆回cysG启动子上游时,厌氧生长期间的转录速率高于有氧生长期间,并且会被亚硝酸盐进一步诱导。这些增加完全依赖于功能性的fnr基因,并且通过S1作图实验表明是由于从依赖Fnr的nirB启动子的转录通读。位于nirB基因N端编码区域内的BamHI位点与位于nirC C端的cysG启动子之间的DNA片段没有启动子活性。