The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription
The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription
复制标题
DOI：
10.1128/jb.179.2.423-429.1997
复制
发表时间：
1997-01-01
影响因子：
3.2
通讯作者：
Reznikoff, WS
中科院分区：
文献类型：
--
作者：
Czarniecki, D;Noel, RJ;Reznikoff, WS
乳糖(Lac)操纵子启动子受分解代谢基因激活子-环AMP复合体(CAP)的正调控，该复合体与位于转录起始点上游61.5bp的DNA结合。在CAP结合位点和核心启动子序列之间是一个13bp的序列(从-38到-50[-45区])。通过分离和鉴定随机多点突变，研究了-45区在确定lac表达的CAP非依赖性水平和在CAP激活过程中的可能作用。只有一小部分突变体对Lac启动子的活性有显著影响。在确实影响表达的突变中，观察到非CAP活性的体内lac启动子活性范围是26倍。最高水平的不依赖于CAP的lac表达(是野生型lac启动子水平的13倍)与-40至-45序列的变化相关，并且需要完整的RNA聚合酶cr亚基才能在体外表达，正如上游DNA识别元件所预期的那样。突变启动子在体内被CAP刺激的能力不同，水平从2倍到22倍的野生型水平不等。只有对CAP的反应性的两倍变化可以归因于直接的DNA序列效应。-40到-45序列依赖的启动子活性增强和CAP对启动子活性的刺激不是相加作用的。突变启动子还表现出其他特征，如与INC P1重叠的新生启动子样活性的激活，在一例中，启动子活性依赖于复制子的变化。
The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site. Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from -38 to -50 [the -45 region]). The possible roles of the -45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations. Only a small percentage of mutants have dramatic effects on lac promoter activity. Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity. The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the -40 to -45 sequence and required an intact RNA polymerase cr subunit for in vitro expression, as expected for an upstream DNA recognition element. Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold. Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects. The -40 to -45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively. The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping Inc P1 and, in one case, replicon-dependent changes in promoter activity.