Transcriptional activation mechanism by the memory effect of single RNA polymerase-promoter complex molecules

转录起始的机制被认为是一个由三个基本步骤组成的序列：RNA聚合酶与启动子之间形成一个复合体(闭合复合体)，与部分熔融的DNA双链形成另一个复合体形成磷酸二酯键(开放复合体和化学反应)，以及RNA聚合酶从与RNA伸长相关的启动子中逃逸(启动子清除)。有一种称为“流产引发”的过程，是一种反复合成和释放寡核苷酸分子的过程，通常被排除在机制的主流之外。尽管目前分离的所有原核和真核核糖核酸聚合酶都在体外观察到了这一过程，但它的作用以及它在体内的存在还不清楚。我们几年来一直在澄清，这种顺序机制不是这样的，它的起始是沿着分支路径进行的，其中一条包含死亡的复合体，被定义为只产生ABOR…的复合体更活跃，没有完整的成绩单。以下是它的特点。1.濒临死亡的复合体和合成全长产物的生产性复合体都是由同一组分的酶分子形成的，分子与启动子DNA的解离消除了它们之间的任何差异。2.证实了这些络合物之间的结构差异。3.在一些启动子中，垂死的复合体被转化为死胡同的复合体，它仍然保留着一个短的转录本，但没有伸长活性。因此，启动途径被分成常规的生产途径和可能导致死胡同的流产途径。4.濒临死亡的复合体的命运要么是作为死胡同的复合体失活，要么是从DNA解离，要么直接转化为生产性复合体，这些反应的速度随启动子的不同而不同。5.影响濒临死亡的复合体命运的因素取决于启动子。为了检查体内分支通路的存在和意义，我们选择了GreA和Greb作为线索。在体外，在高浓度的起始核苷三磷酸存在的情况下，在LP_ral启动子上，这些因素促进了垂死的复合体向生产性复合体的转化。如果体内存在分支机制，GRE因子的缺失应该会导致启动子的生产性转录减少，在这种情况下，垂死的复合体对这些因子很敏感。我们构建了大肠杆菌的双干扰株DgreA DgreB，然后从突变株中发现转录水平低于亲本株的10个基因中随机选择了10个基因。最后，其中三个基因的启动子atpC(UNCC)、CSPA和rpsA通过了进一步的常规测试，证实它们在由纯化成分组成的重组转录系统中显示了一条分支起始途径。这些结果证明，分支起始途径在体内是存在的，并通过调节进入每个分支的聚合酶-启动子复合体的比例来调节某些启动子的转录起始。观察到对Greb水平的测定是结构性的。在整个生长阶段，GREA的水平保持不变，对培养介质的丰富性没有太大反应。然而，在有氧条件下，它下降到一半，这表明一些基因受到GREA的调控。因此，对涉及GREA的蛋白质水平做出反应的调节回路，即分支通路机制，在细胞中发挥作用。较少

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 eukaryotic 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