Immunoaffinity Purification of the Human Multisubunit Transcription Factor IIH

Abstract

A procedure to immunoaffinity purify the human transcription factor IIH (TFIIH) was developed using a monoclonal antibody that recognizes an epitope in ERCC3 (XPB), the largest subunit of TFIIH. The epitope recognized by the monoclonal antibody was mapped to 20 amino acids. A peptide containing the epitope was capable of displacing TFIIH from an immunoaffinity column containing the monoclonal antibody. The immunoaffinity purification procedure described allows a simple and efficient method to purify both the "core" and "holo" TFIIH complexes.Initiation of transcription by RNA polymerase II (RNAPII) is a complex process requiring the assistance of five factors known as the general transcription factors (GTFs). These are transcription factor (TF) 1 IIB, TFIID, TFIIE, TFIIF, and TFIIH (1, 2). TFIID is a multisubunit complex that includes the TATA binding protein (TBP) (3). TBP, in its recombinant form or as part of the TFIID complex, recognizes the TATA motif present in many genes and can nucleate the formation of transcriptioncompetent complexes (4). The TBP (TFIID)-TATA complex provides the foundation for the entry of TFIIB, which is then followed by the entry of RNA polymerase II. RNA polymerase II is escorted to the promoter by TFIIF (5). The resulting DNAprotein complex, TBPolF, contains RNA polymerase II stably associated with the promoter (2). This complex, however, is unique with respect to complexes formed with other RNA polymerases since the formation of an RNA polymerase II transcription-competent complex requires the function of two other GTFs, TFIIE and TFIIH (6).TFIIE is a tetrameric factor composed of two subunits, p56 and p34 (7,8). TFIIE interacts with RNA polymerase II (9) and TFIIH (10,11). It is currently unknown whether TFIIE enters the transcription complex with RNA polymerase or in a subsequent step with TFIIH. However, it is clear that the association of TFIIE with the TBPolF complex is necessary for the entry of TFIIH to the transcription complex (6). Recent studies have suggested an alternative model for the formation of transcription competent complexes. These studies suggest that the GTFs and RNA polymerase II are preassembled in an RNA polymerase holoenzyme complex (12)(13)(14).TFIIH also known as BTF2 was initially purified as a factor required to reconstitute accurate transcription by RNA polymerase II in vitro (6,15). BTF2 was originally presumed to be TFIIE (16). However, the availability of antibodies against a subunit of TFIIE (17) revealed that BTF2 and TFIIE are distinct (18). Furthermore, antibodies generated against the 62-kDa subunit of BTF2 revealed that BTF2 and TFIIH are the same factor (18,19). Additionally, an activity isolated from rat liver extracts, referred to as factor ␦, contained a subunit composition and properties similar to human TFIIH (20). Purification of TFIIH demonstrated that it is a multisubunit factor composed of nine polypeptides (6,15,20). The studies with the rat liver-derived factor were the first to demonstrate that factor ␦ (TFIIH) copurified wit...

Nucleolin Is Required for RNA Polymerase I Transcription In Vivo

“…The purification of RNA polymerase II and transcription factor IIH (TFIIH) from mammalian cell nuclear extract and of TATA-binding protein (TBP), TFIIB, TFIIE, and TFIIF synthesized in Escherichia coli was done as described previously (47). Core histones, RSF, and FACT were purified from HeLa cell nuclei by following previously described protocols (40,57,79). A chimeric activator (GAL4-AD) composed of a FLAG-tagged GAL4 DNA-binding domain fused to the activation domain of c-Myc was synthesized in Hi-5 insect cells and purified by immunoaffinity chromatography as described previously (50).…”

“…Biochemical studies have established that members of the SWI/SNF family use ATP hydrolysis to alter the conformation of histones within a nucleosome (32,36,42,74). Members of the ISWI family, which all possess a homolog of the Drosophila ISWI protein, have been identified in many organisms and use energy from ATP hydrolysis to slide nucleosomes translationally along DNA (33,40,41,63,84).…”

The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7

“…TFIIH was isolated by an immunoaffinity purification method by use of monoclonal antibodies against ERCC3 (LeRoy et al 1998). A silver staining of a polyacrylamide gel containing a representative TFIIH used in the experiments described below is shown in Figure 2b (see Fig.…”

“…Immunoaffinity purification of TFIIH with ERCC3 monoclonal antibodies was performed as described (LeRoy et al 1998). In short, proteins from the second step of purification of TFIIH, the DEAE-cellulose bound fraction, were incubated with protein A-immobilized anti-ERCC3 monoclonal antibodies.…”

“…This trimeric complex is known as the cdk-activation kinase (CAK) complex and was initially isolated as an activator of different CDKs (Fisher and Morgan 1994). Holo-TFIIH is necessary for transcription activity (Svejstrup et al 1995;Drapkin et al 1996;Reardon et al 1996;Marinoni et al 1997;LeRoy et al 1998), yet the CDK7-kinase activity is dispensable for transcription of some genes in vitro (Akoulitchev et al 1995;Makela et al 1995). The regulation of CDK7 (CAK) activity has been the subject of extensive studies (Shuttleworth et al 1990;Labbe et al 1994;Tassan et al 1994;Fisher et al 1995;Martinez et al 1997).…”