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In cells, alongside organelles with lipid membrane, exist organelles without membrane. These dynamic membranelles organelles are formed by liquid-liquid phase separation (LLPS) mechanism; they serve as microreactors, increasing a local concentration of components and accelerate the reaction. In eukaryotic cells, the complex responsible for transcribing of protein-coding is RNA polymerase II (RNAPII). This enzyme clusters into droplets at transcribed genes via its C-terminal, disordered domain. The molecular mechanism of RNAPII clustering remains unknown.
Here, we present a unique technique of co-expression and purification of the complex of human RNAPII from Hi5 cells. Three subunits of RNAPII were fused with specific affinity tags, which allows us to purify protein using affinity chromatography. Because preparation of RNAPII in suitable concentrations from Hi5 cells seems to be problematic, we simultaneously work on a protocol to purify RNAPII from HEK293 cells.
The purified RNAPII will allow us to study the process of phase separation of human RNAPII into droplets in vitro by a combination of structural and functional studies.