CHARACTERIZATION OF THE OXYGEN-DEPENDENT PROMOTER OF THE VITREOSCILLA HEMOGLOBIN GENE IN ESCHERICHIA-COLI
CHARACTERIZATION OF THE OXYGEN-DEPENDENT PROMOTER OF THE VITREOSCILLA HEMOGLOBIN GENE IN ESCHERICHIA-COLI
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DOI：
10.1128/jb.171.11.5995-6004.1989
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发表时间：
1989-11-01
影响因子：
3.2
通讯作者：
BAILEY, JE
中科院分区：
文献类型：
--
作者：
KHOSLA, C;BAILEY, JE
编码玻璃体血红蛋白（VHb）分子的基因已在大肠杆菌中克隆并功能性表达。通过使用质粒编码的基因以及单拷贝整合体，氧依赖性VHb基因（VHb）启动子在E.杆菌该启动子在微需氧条件下（溶解氧水平低于2%空气饱和度）被最大程度地诱导。mRNA水平的直接分析以及与lacZ的基因融合的使用表明，氧依赖性调节发生在转录水平。在缺氧条件下，转录活性显著降低，表明在E.杆菌引物延伸分析被用来确定存在两个重叠的启动子内的结构VHb基因上游的150个碱基对的区域。这两个启动子的氧依赖性活性是定性相似的，这表明存在一个共同的机制，通过该机制，可用的氧浓度影响两个启动子的表达。crp和cya突变体的启动子活性分析表明，环AMP和分解代谢物激活蛋白的启动子的全部活性所需的。VHb启动子含有一个与E. colilac启动子。
The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of significant homology to the catabolite activator protein-binding site near the E. coli lac promoter.