Method for searching bacterial promoters based on the mechanism of transcription initiation

转录起始的机制被认为是一系列的三个基本步骤：RNA聚合酶和启动子之间形成复合体（封闭复合体），另一个复合体与部分融化的DNA双链形成磷酸二酯键（开放复合体和化学反应），RNA聚合酶从与RNA延伸过程相关的启动子中逃逸（启动子清除）。有一个被称为“流产起始”的过程，即寡rna分子的迭代合成和释放，经常被排除在主流机制之外。尽管这一过程在体外已被观察到，迄今分离出的所有原核和真核RNA聚合酶，其作用以及在体内的发生尚不清楚。几年来，我们一直在澄清，这种顺序机制并非如此，起始遵循分支途径，其中一个包含垂死复合体，被定义为只产生流产的复合体……更多的，没有全长转录本。以下是其特点。1.垂死的复合体，以及合成全长产物的生产复合体，都是由相同的酶分子的均匀部分形成的，分子与启动子DNA的解离消除了它们之间的任何差异。2.这些配合物之间的结构差异已被证明。3.在一些启动子中，垂死复合体转化为死端复合体，仍然保留短转录本，但没有延伸活性。因此，起始途径分为常规生产途径和可能导致死胡同的流产途径。4.垂死复合体的命运要么是作为死端复合体失活，要么与DNA分离，要么直接转化为生产复合体，这些反应的速率随启动子的不同而变化。5.有一些因素以某种方式影响垂死复合体的命运，这取决于启动子。为了检验分支通路在体内的存在及其意义，我们选择了GreA和GreB作为线索。在lP_RAL启动子处，在体外高浓度启动三磷酸核苷存在的情况下，这些因子促进了濒死复合体向生产复合体的转化。如果分支机制在体内存在，那么缺乏Gre因子将导致启动子的高产转录减少，而濒死复合体对这些因子敏感。我们构建了大肠杆菌的双干扰物DgreA DgreB，然后从突变菌株中随机选择10个转录本水平低于亲本菌株greA^+greB^+的基因。最后，其中三个基因atpC （uncC）、cspA和rpsA的启动子通过了进一步的常规测试，证实它们在由纯化组分组成的重组转录系统中表现出分支起始途径。结果证明，分支起始途径在体内存在，并通过调节进入该途径各分支的聚合酶启动子复合物的比例，用于调控某些启动子的转录起始。观察到GreB水平的测定是本构性的。在整个生长阶段，GreA的水平保持不变，并且对培养基的丰富度没有太大的反应。然而，在有氧条件下，它减少了一半，这表明一些基因是由GreA调节的。因此，对涉及GreA的蛋白水平作出反应的调控回路，即分支通路机制，在细胞中起作用。少

The mechanism of transcription initiation has been assumed to be a sequence of three essential steps: formation of a complex between RNA polymerase and a promoter (closed complex), formation of another complex with partially melted DNA duplex to form phosphodiester bonds (open complex and chemical reaction), and escape of RNA polymerase from the promoter associated with the progress of RNA elongation (promoter clearance). There is a process called "abortive initiation", an iterative synthesis and release of oligo-RNA molecules, and is often excluded from the mainstream of the mechanism. Altough this process has been observed in vitro with all prokaryotic and erkaryotic RNA polymerases so far isolated, its role as well as its occurrence in vivo has been unknown.We have been clarifying for several years that this sequential mechanism is not the case and the initiation follows branched pathways, one of which contains the moribund complex, being defined as a complex that produces only abor … More tive and no full-length transcripts. Followings are its characteristics. 1.The moribund complex, as well as the productive complex that synthesizes full-length product, are formed from the same homogeneous fraction of enzyme molecules, and dissociation of the molecules from the promoter DNA cancels any difference between them. 2.Structural differences between these complexes have been demonstrated. 3.At some promoters, a moribund complex is converted into a dead-end complex that still retains a short transcript but has no elongation activity. Therefore, the initiation pathway is branched into the conventional productive pathway and the abortive pathway that can lead towards a dead end. 4.The fates of a moribund complex are either inactivation as a dead-end complex, dissociation from the DNA, or direct conversion into a productive complex, and the rates of these reactions vary with the promoter. 5.There are factors that affect the fate of the moribund complex in a manner that depends on the promoter.To examine the existence and significance of the branched pathway in vivo, we selected GreA and GreB for clues. At the lP_RAL promoter these factors enhance conversion of the moribund complex into the productive one, in the presence of high concentrations of initiating nucleoside triphosphate in vitro. If the branched mechanism exists in vivo, absence of the Gre factors should result in reduction of productive transcription from promoters at which the moribund complex is susceptible to these factors. We constructed a double-disruptant of E.coli, DgreA DgreB, and then arbitrarily selected 10 genes from among those whose levels of transcripts in the mutant strain were found to be lower than those in the parental greA^+greB^+ strain. Finally, the promoter for three of these genes, atpC (uncC), cspA, and rpsA, passed a further conventional test which confirmed that they displayed a branched initiation pathway in a reconstituted transcription system composed of purified components. The results obtained prove that the branched initiation pathway exists in vivo and is utilized in regulation of transcription initiation from some promoters, through modulation of the fraction of polymerase-promoter complexes entering each branch of the pathway. The determination of the level of GreB was observed to be constitutive. The level of GreA remained the same through the growth phase, and did not respond much to the richness of the culture media. However, it decreased into half in aerobic conditions, indicating that some genes are regulated by GreA. Therefore, the regulatory circuit responding to the levels of proteins involving GreA, namely the branched pathway mechanism, is working in cells. Less