Sharp J A, Platt T
J Biol Chem. 1984 Feb 25;259(4):2268-73.
We have investigated the specific DNA and RNA requirements for rho-dependent transcription termination in vitro. As a model, we have used templates containing the rho-dependent terminator of the Escherichia coli trp operon, trp t'. Templates containing the trp t' region direct specific rho-dependent termination in vitro, with concomitant stimulation of the rho NTPase activity, and deletion of the trp t' region results in templates that do not induce rho-dependent termination or rho NTPase activity. Addition of ribonuclease T1 to transcription reactions specifically eliminated transcription termination and rho NTPase activity. These results demonstrate the requirement for a specific RNA component within the trp t' transcript necessary for NTPase activation and rho-dependent transcription termination. Active transcription is not a prerequisite for rho NTPase activation; trp t' RNA (rho-terminated transcripts) and read-through transcripts, which contain the trp t' region, activated the rho NTPase when rho was added after inhibition of transcription. As is true for synthetic polynucleotides known to activate the rho NTPase, the trp t' region has few G residues. This reduces the potential for the formation of stable secondary structures in the RNA transcript, and may be one determinant of sites specifying rho-dependent termination of transcription. The implications of this are discussed in the light of the lack of significant sequence homologies between rho-dependent transcription termination sites.
我们已经在体外研究了rho依赖性转录终止所需的特定DNA和RNA条件。作为一个模型，我们使用了含有大肠杆菌色氨酸操纵子rho依赖性终止子trp t'的模板。含有trp t'区域的模板在体外指导特定的rho依赖性终止，同时刺激rho NTPase活性，而删除trp t'区域会导致模板不诱导rho依赖性终止或rho NTPase活性。向转录反应中添加核糖核酸酶T1可特异性消除转录终止和rho NTPase活性。这些结果证明了trp t'转录本中存在特定RNA成分对于NTPase激活和rho依赖性转录终止是必需的。活跃转录不是rho NTPase激活的先决条件；trp t' RNA（rho终止的转录本）和包含trp t'区域的通读转录本在转录抑制后添加rho时会激活rho NTPase。正如已知能激活rho NTPase的合成多核苷酸一样，trp t'区域的G残基很少。这降低了RNA转录本中形成稳定二级结构的可能性，并且可能是指定rho依赖性转录终止位点的一个决定因素。鉴于rho依赖性转录终止位点之间缺乏显著的序列同源性，对此进行了讨论。
