Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase.
Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase.
复制标题
Mu C 蛋白激活噬菌体 Mu lys 启动子需要大肠杆菌 RNA 聚合酶的 sigma 70 亚基。
DOI：
10.1128/jb.172.3.1424-1429.1990
复制
发表时间：
1990
影响因子：
3.2
通讯作者：
Howe,MM
中科院分区：
文献类型：
--
作者：
Margolin,W;Howe,MM
噬菌体Mu C蛋白是中间操纵子的产物，是激活四个Mu晚期启动子所必需的。为了解决其作用机制，我们从含有C基因的质粒中过量生产了约16.5千道尔顿的C蛋白，该基因受噬菌体T7启动子和核糖体结合位点的控制。高度富集大肠杆菌RNA聚合酶（E sigma 70）并由过量生产菌株制成的蛋白质级分能够在体外激活来自tac启动子（Ptac）和Mu late启动子Plys的转录。Plys在体内和体外的行为是相似的;在两种条件下，转录是C依赖性的，并且RNA 5'末端是相同的。当将抗σ 70抗体添加到含有Ptac和Plys模板的C依赖性转录反应中时，来自两种启动子的转录被抑制;通过添加过量的E σ 70来恢复转录。这一结果表明，C依赖性激活的Plys需要σ 70。进一步的支持证据由重构实验提供，其中含有C的E sigma 70耗尽的级分不能激活Plys的转录，除非加入纯化的sigma 70和核心聚合酶。这些结果强烈表明，C不是一个新的西格玛因子，但作为激活剂E西格玛70依赖性转录。
Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters. To address its mechanism of action, we overproduced the approximately 16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Ptac) and a Mu late promoter, Plys. The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical. When anti-sigma 70 antibody was added to C-dependent transcription reactions containing both Ptac and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess E sigma 70. This result suggests that C-dependent activation of Plys requires sigma 70. Further supporting evidence was provided by a reconstitution experiment in which an E sigma 70-depleted fraction containing C was unable to activate transcription from Plys unless both purified sigma 70 and core polymerase were added. These results strongly suggest that C is not a new sigma factor but acts as an activator for E sigma 70-dependent transcription.