Isolation & Characterization of Transcription Factories

我们如何设想体外转录？ 大多数体外转录研究都是用纯化的RNA聚合酶进行的，该聚合酶转录相对较短的DNA片段。常识告诉我们，在这个系统中，RNA聚合酶应该像一个小的球形物体，以每秒20-30个碱基对的速度沿着DNA移动（和旋转）。这种观点完全来自于我们对DNA的看法，即DNA是一根长而粗的杆，而RNA聚合酶是一个小珠子，可以沿着杆沿着自由移动。 我们的假设可能是错误的吗？ 现代研究以几种方式挑战RNA聚合酶的体外感知。近年来所做的大量工作揭示了RNA聚合酶是许多不同蛋白质（转录因子）的庞大复合物，这些蛋白质来来去去，而聚合酶则在基因中移动。此外，信使RNA在核糖体上的翻译、RNA剪接和加工通常共转录地发生，使得数百种核糖体和mRNA加工蛋白质在体内被束缚到RNA聚合酶。 多年前，我们发现通过蛋白质中的亲和标签附着于聚合物珠的RNA聚合酶在转录循环的所有步骤中正常进行，包括启动子处的起始、RNA的延伸和转录的终止（Kashlev，1993; Sidorenkov，1998; Kireeva，2000; Komissarova，1998; 2002）。在该系统中，通过含有核小体的DNA的转录也不受影响（Kireeva，2002）。当酶被固定时，转录不能通过聚合酶沿着DNA的移动和旋转进行。转录是如何发生的？通过观察完整的真核细胞中转录是如何发生的以及在哪里发生的，这一争论得到了缓解（Peter Cook，2001）。这些实验表明，体内RNA合成发生在固定的“转录工厂”中，该工厂包含一个固定的RNA聚合酶分子池，该分子连接到细胞核的固体元件上（Peter Cook，2000）。转录被认为是在这些工厂中通过将模板穿过固定化的酶而发生的，并且它可能涉及将基因暂时募集到这些固定位点。转录工厂可以用电子或荧光显微镜观察到，但从来没有用生物化学方法研究过。 转录工厂：从体内可视化到体外纯化和重建。 该项目涉及转录工厂的分离和生物化学表征，在独特的酵母（S。酿酒酵母）或细菌（E. coli）基因。对于工厂的生化分离，我们开发了一种新技术，通过专门设计的转录路障来“停止”和稳定细胞中的工厂，然后使用特殊的标签纯化工厂，将其引入其中一个组件。在进一步发展后，该项目将包括比较在体内不同酵母和细菌基因上组装的工厂，以及在体外重建“最小”转录工厂。

How do we envisage transcription in vitro? Most of the in vitro studies of transcription are performed with purified RNA polymerase that transcribes relatively short DNA fragments. Common sense tells us that in this system RNA polymerase should act as a small spherical object that moves (and rotates) along DNA with a rate of 20-30 base pairs per second. This view derives entirely from our perception of DNA as a long bulky rod and RNA polymerase as a small bead freely moving along the rod. May we be wrong in our assumption? Modern research challenges the in vitro perception of RNA polymerase in several ways. The extensive work done in recent years revealed RNA polymerase as a bulky composite of many different proteins (transcription factors) which come and go, while the polymerase moves across the gene. Moreover, translation of messenger RNA at ribosomes, RNA splicing and processing often occur co-transcriptionally making hundreds of ribosomal and mRNA processing proteins tethered to RNA polymerase in vivo. Years ago, we found that RNA polymerase, which was attached to the polymeric beads through the affinity tag in the protein, performed normally in all steps of the transcription cycle including initiation at promoter, elongation of RNA and termination of transcription (Kashlev, 1993; Sidorenkov, 1998; Kireeva, 2000; Komissarova, 1998; 2002). Transcription through DNA containing nucleosomes was also not affected in this system (Kireeva, 2002). When the enzyme was immobilized, transcription could not proceed by movement and rotation of the polymerase along the DNA. How did the transcription occur? This controversy was relieved by the observation of how and where transcription occurs in the intact eukaryotic cells (Peter Cook, 2001). These experiments showed that RNA synthesis in vivo takes place in the stationary "transcription factories", containing a pool of immobilized RNA polymerase molecules attached to the solid elements of the nucleus (Peter Cook, 2000). Transcription is believed to occur in these factories by threading the template through the immobilized enzyme, and it may involve temporary recruitment of the genes to these immobile sites. The transcription factories could be visualized with the electron or fluorescent microscopy, but they were never approached biochemically. Transcription Factory: from visualization in vivo to purification and reconstitution in vitro. The project involves isolation and biochemical characterization of the transcription factory, assembled at the unique yeast (S. cerevisiae) or bacterial (E. coli) gene in vivo. For the biochemical isolation of the factory, we developed a novel technology for "halting" and stabilizing the factory in the cell by specially designed transcription roadblocks, followed by purification of the factory using a special tag, introduced to one of its components. Upon further development, the project will include a comparison of the factories assembled on different yeast and bacterial genes in vivo, and reconstitution of the "minimal" transcription factory in vitro.