title: Improved method for soluble expression and rapid purification of yeast TFIIA.
abstract: 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/Rhvalue 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.
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan; Department of Materials Structure Science, School of High Energy Accelerator Science, The Graduate University of Advanced Studies (Soken-dai), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. Electronic address: naruhiko.adachi@kek.jp.
authors.forename: Naruhiko
authors.initials: N
authors.lastname: Adachi
authors.affiliation: Department of Materials Structure Science, School of High Energy Accelerator Science, The Graduate University of Advanced Studies (Soken-dai), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. Electronic address: aizawak@post.kek.jp.
authors.forename: Kyohei
authors.initials: K
authors.lastname: Aizawa
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. Electronic address: yukay@post.kek.jp.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kratzer
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. Electronic address: shinya.saijo@kek.jp.
authors.forename: Shinya
authors.initials: S
authors.lastname: Saijo
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan; Department of Materials Structure Science, School of High Energy Accelerator Science, The Graduate University of Advanced Studies (Soken-dai), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. Electronic address: nobutaka.shimizu@kek.jp.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Shimizu
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan; Department of Materials Structure Science, School of High Energy Accelerator Science, The Graduate University of Advanced Studies (Soken-dai), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. Electronic address: toshiya.senda@kek.jp.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Senda
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D035561:Transcription Factor TFIIA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C441519:TOA1 protein, S cerevisiae; C441520:TOA2 protein, S cerevisiae; D035561:Transcription Factor TFIIA
keywords: Polyprotein strategy; Purification; TFIIA; Transcription