Improved method for soluble expression and rapid purification of yeast TFIIA

- We developed an improved method for purifying TFIIA.
- The subunits of TFIIA, TOA1 and TOA2, were expressed by a polyprotein strategy.
- Physicochemical analyses showed a heterodimer formation of purified TOA1 and TOA2.
- The intrinsically disordered region of TFIIA might not adopt an extended structure.
- A broad range of researchers will be able to obtain TFIIA via our improved method.

In vitro transcription systems have been utilized to elucidate detailed mechanisms of transcription. Purified RNA polymerase II (pol II) and general transcription factors (GTFs) are required for the in vitro reconstitution of eukaryotic transcription systems. Among GTFs, TFIID and TFIIA play critical roles in the early stage of transcription initiation; TFIID first binds to the DNA in transcription initiation and TFIIA regulates TFIID's DNA binding activity. Despite the important roles of TFIIA, the time-consuming steps required to purify it, such as denaturing and refolding, have hampered the preparation of in vitro transcription systems. Here, we report an improved method for soluble expression and rapid purification of yeast TFIIA. The subunits of TFIIA, TOA1 and TOA2, were bacterially expressed as fusion proteins in soluble form, then processed by the PreScission protease and co-purified. TFIIA's heterodimer formation was confirmed by size exclusion chromatography−multiangle light scattering (SEC−MALS). The hydrodynamic radius (Rh) and radius of gyration (Rg) were measured by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), respectively. The Rg/Rh value implied that the intrinsically disordered region of TOA1 might not have an extended structure in solution. Our improved method provides highly purified TFIIA of sufficient quality for biochemical, biophysical, and structural analyses of eukaryotic transcription systems.