Role of the single-molecule memory in transcriptional regulation

几十年来，原核生物早期转录的机制被认为是一个由三个基本步骤组成的序列：开放复合体的形成，化学反应的启动，RNA聚合酶随着RNA的伸长而逃逸出启动子。然而，到目前为止分离到的所有原核和真核RNA聚合酶都具有“流产起始”功能，即在体外反复合成和释放寡聚RNA分子，但其作用尚未确定。作为基于这些开创性工作的最小模型，流产转录本被认为是全长转录本的不成功前体，因此流产合成被认为是序列模型中启动子清除的一个步骤。我们检验了这一假设，发现流产转录本的主要来源是不能产生成熟转录本的复合体。我们将这个复数命名为垂死的复数，并证明了它是由齐次的Pre…形成的进一步分离了RNA聚合酶作为产物复合体，即利用RNA聚合酶启动子复合体的构象作为单分子记忆的转录起始分支途径。我们最终证明了该机制作为一种转录启动的调节机制在大肠杆菌中起作用，当两种构象在RNA伸长的时间尺度上不可逆时，该机制有其生理后果。这是lambda PR和lacUV5启动子的情况，而T7A1和rrnBP1启动子的情况是可逆的。我们发现作为延伸因子的GREA和GREB使一些启动子更具可逆性，并通过从长时间死亡的复合体到迅速消失的生殖性起始复合体的净转化来激活转录。这些因素被发现作用于ATP操纵子的主要启动子cspAP1、rpsAP1、rpsAP3和Labbda PR，证明分支途径在体内起作用。这些起始/延长因子的明显特征不是与DNA结合，而是与RNA聚合酶的次要通道Dks A结合，在严格控制下放大ppGpp的作用。这意味着通过控制可逆性对启动的调控可以与ppGpp和DksA的调控竞争，找到了两种调控机制的串扰。较少

For several decades, the mechanism of in the early transcription in prokaryotes has been assumed to be a sequence of three essential steps : formation of open complex, initiation of the chemical reaction, and escape of the RNA polymerase from the promoter along with RNA elongation. However, all prokaryotic and eukaryotic RNA polymerases so far isolated have been known to perform "abortive initiation", which is an iterative synthesis and release of oligo-RNA molecules in vitro but its role has not been identified. As a minimal model based on these pioneering works, abortive transcripts were assumed to be unsuccessful precursor of the full-length transcripts, and thus abortive synthesis has been supposed to be a step of the promoter clearance in a sequential model. We examined this hypothesis and found that the major source of abortive transcripts is a complex that cannot produce mature transcript. We named this complex moribund complex and proved that it is formed from a homogeneous pre … More paration of RNA polymerase as the productive complex, namely the branched pathway of transcription initiation using the conformations of RNA polymerase promoter complex as a single-molecular memory. We finally proved that this mechanism is working as a regulatory mechanism of transcription initiation in E.coli.This mechanism has its physiological consequence when the two conformations are irreversible in the time scale of RNA elongation. This is the case of lambda PR and lacUV5 promoters and a reversible cases are found for T7A1 and rrnBP1 promoters. We found GreA and GreB, which have been found as elongation factors, make some promoters more reversible and activate transcription by the net conversion from long-lived moribund complex into rapdly disappearing productive initiation complex. The factors were found to work on the major promoter of atp operon, cspAP1, rpsAP1, rpsAP3, and labbda PR, proving that the branched pathway is working in vivo. The distinct feature of these initiation/elongation factors bind not to DNA but to the minor channel of RNA polymerase as DksA that amplify the action of ppGpp in stringent control. This means the regulation of initiation by controlling the reversibility can compete with the regulation by ppGpp and DksA, finding a cross-talk of two regulatory mechanisms. Less