Isolation and Characterisation of Transcription Factorie

现代的真核转录概念认为，核内RNA的合成是在固定的“转录工厂”(转录工厂)中进行的，转录工厂包含固定在固体核支架上的Pol II分子池。最近的报道还表明，原核生物中也可能存在转录因子。固相转录可能包括暂时将基因重新招募到工厂，在工厂中，转录是通过将模板穿过固定化的酶来进行的。转铁蛋白的蛋白质组成极其复杂且高度动态化。研究表明，转录因子可能含有RNA聚合酶、转录延伸因子、mRNA修饰、加工/剪接机制和核翻译装置的组成部分。转录工厂可以用EM或荧光显微镜观察，但从来没有人用生物化学方法接近过它们。这个项目涉及对转录因子的生化表征，该转录因子组装在染色体或质粒中的报告基因上。我们开发了一种新的技术，通过特殊设计的转录障碍(Lac抑制物，结合到操纵子DNA)，在体内“停止”和稳定Tf，然后使用亲和标签进行Tf纯化，引入到Tf的一个组成部分。在进一步的发展中，该项目将涉及对装配在不同酵母基因上的TF进行比较，或者对工厂组装在同一基因上但位于不同染色体位置的TF进行比较。

The modern concept of eukaryotic transcription suggests that RNA synthesis in nucleus takes place in the stationary "transcription factories" (TFs), containing a pool of immobilized Pol II molecules attached to the solid nuclear scaffold. Most recent reports also suggest that TFs may exist in prokaryotes as well. The solid-phase transcription may involve temporary recruitment of the genes to the factory where transcription is proposed to occur by threading the template through the immobilized enzyme. The protein composition of the TFs is extremely complex and highly dynamic. It has been shown that the TFs may contain RNA polymerase, transcription elongation factors, mRNA modification, processing/splicing machinery, and components of nuclear translation apparatus. The transcription factories can be visualized with EM or fluorescent microscopy, but they have never been approached biochemically. This project involves biochemical characterization of the TF, assembled at a reporter gene in a chromosome or on a plasmid. We developed a novel technology for "halting" and stabilizing the TF in vivo by specially designed transcription roadblocks (lac repressor, bound to the operator DNA), followed by the TF purification using an affinity tag, introduced to one of the TF components. Upon further development, the project will involve a comparison of the TFs assembled on different yeast genes, or the factory assembled on the same gene, but in different chromosome locations.