大肠杆菌中galE转录暂停调控下游ρ依赖型转录终止的分子机制

The first step of gene expression, during which a particular segment of DNA is copied into RNA by RNA polymerase is called transcription. Instead of being a smooth, continuous process, during transcription the RNA polymerase is prone to pause at special sites whileas the conformation of RNA polymerase active center changes into an unproductive state. Transcriptional pausing, whose function is thought to regulate downstream gene expression, is affected by various factors such as nucleic acid sequences around the pausing site, secondary structure of the newly formed RNA near the active center, transcription elongation factors and ribosomes, etc. While transcriptional pausing occurs frequently in E. coli, the mechanisms and effects are poorly understood and more thorough research is needed..Galactose operon contains four structural genes, namely galE, galT, galK and galM, which jointly encode enzymes for galactose degradation. There are four ρ-dependent termination sites located at the 3’ends of each gene. In our previous study, we observed for the first time that there are several transcription pausing sites in the coding region of galE, the first structural gene in galactose operon. Furthermore, these pausing sites were identified as rut sites (Rho utilization sites), i.e., binding sites for ρ factor, a protein involved in transcription termination. It was recently reported that ρ factor associates with RNA polymerase during the whole transcription cycle, while it promotes transcription termination only at termination sites. However, how ρ factor binds to RNA polymerase remains controversial. On the basis of previous work, we propose that ρ factor binds to the paused RNA polymerase through the rut sites, subsequently promoting ρ-dependent termination at downstream genes, and pausing provides time for ρ factor binding. .The phenomenon that transcriptional pausing occurs at rut sites of the first structural gene is also observed in several other operons, indicating that it is not occasional. In this research, single-round in vitro transcription, ChIP, 3’RACE combined with primer extension will be applied to illustrate the mechanism of galE pausing on ρ factor association and downstream gene expression, which is expected to expand our understanding of transcription regulation. This project is also instructive in designing functional gene modules in synthetic biology field as well.

由RNA聚合酶(RNAP)介导的转录不是持续进行的，在特定条件下会暂停。E. coli中已知的转录暂停均由DNA序列引起，具有调控下游基因表达的功能。申请人首次在E. coli半乳糖操纵子的galE编码区观察到了转录暂停现象，它不含已知的转录暂停保守序列，并且暂停位点附近有转录终止因子ρ的结合序列(rut序列)，申请人在其它基因中也观察到该规律，因此推测它是一种与ρ因子相关的新的普遍性的转录暂停形式。.本研究拟通过体外单循环转录、ChIP、3’RACE结合引物延伸等方法揭示galE转录暂停调控下游基因表达的分子机制。包括转录暂停如何为ρ因子的结合提供机会与时间，ρ因子通过rut序列与RNAP结合后，怎样调节下游ρ依赖型的转录终止等。.本研究以一种新的转录暂停为研究对象，有望揭示新的转录调控机制，并发现ρ因子与转录暂停相关的新功能，该研究对合成生物学中功能基因的设计具有重要指导意义。

转录是基因表达的一个至为重要的步骤。转录暂停与转录终止紧密相关，它们对基因表达的调控至关重要，然而目前的研究多集中于转录起始阶段，原核生物中转录暂停的研究还相对较少。大肠杆菌拥有两种类型的终止子，内在型转录终止子 (Rho-independent terminator, RIT)及Rho依赖型转录终止子 (Rho-dependent terminator, RDT)。大肠杆菌半乳糖操纵子参与半乳糖分解，其转录终止机理还未知。.在本研究中，我们对其的转录终止机理进行了深入研究。证明了RIT及RDT均发挥转录终止的作用。然而半乳糖操纵子的mM1转录本仅有一个3’末端，位于+4313。RIT的转录终止位点在+4315，RDT的转录终止位点在+4409，因此+4313并不是转录终止的原始位点。我们发现+4313由核酸外切酶RNase II以及核酸内切酶RNase E加工形成。发卡结构抑制RNase II的3’至5’外切酶活性，从而形成稳定的RNA 3’末端。 我们还发现翻译抑制转录终止。我们将galM翻译终止位点TAA突变为密码子AAA，发现RIT及RDT均受到翻译的抑制。转录与翻译紧密偶联时，停留于翻译终止位点的核糖体抑制mRNA发卡结构的形成，抑制RIT。在翻译终止位点核糖体解离后转录与翻译解偶联，RNA聚合酶继续转录至下游，在RDT处发生转录终止。最后，我们对大肠杆菌染色体上850个操纵子下游的转录终止子进行了预测，发现有73% 的RIT与翻译终止子的距离小于30个碱基，暗示着大部分RIT均被翻译抑制。RDT与翻译终止子的距离分布呈随机性，并未表现出特别的规律。有关RIT与RDT转录终止子分布规律的原因还有待进一步研究。.综上所述，本研究的结论主要有两点：第一，RNA 3’末端的产生受到核酸外切酶剪切，此现象在原核生物中普遍存在。第二，不仅半乳糖操纵子，大肠杆菌的大部分操纵子中，RIT受到翻译的抑制。在真核生物中转录终止的机理尚不明确，已知成熟转录本的形成也受到核酸内切酶及外切酶的剪切。因此原核生物与真核生物成转录本的形成机理相似。此研究进一步完善了转录终止的调控机制，为合成生物学中功能基因的设计提供了理论依据。深入研究其调控机理对于找到新的药物靶标、开发新型抗生素具有重大意义。