Isolation and Characterisation of Transcription Factorie

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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.