Facile and scalable expression and purification of transcription factor IIH (TFIIH) core complex.

Transcription factor IIH (TFIIH) plays essential roles in both the initiation of RNA Polymerase II-mediated transcription and the Nucleotide Excision Repair (NER) pathway in eukaryotes. In NER, the 7-subunit TFIIH Core sub-complex is responsible for the opening and extension of the DNA bubble created at the lesion site, utilizing the molecular motors XPB and XPD. Mutations in Core subunits are associated with a series of severe autosomal recessive disorders characterised by symptoms such as mild-to-extreme photosensitivity, premature ageing, physical and neurological anomalies, and in some cases an increased susceptibility to cancer. Although TFIIH Core has been successfully obtained in the past, the process has always remained challenging and laborious, involving many steps that severely hindered the amount of pure, active complex obtained. This has limited biochemical and functional studies of the NER process. Here we describe improved and simplified processes for the cloning, expression and purification of the 7-subunit TFIIH Core sub-complex. The combined use of auto-cleavable 2A-like sequences derived from the Foot-and-Mouth Disease Virus (FMDV) and the MultiBac™ cloning system, a powerful baculoviral expression vector specifically conceived for the obtaining of multi-subunit eukaryotic complexes, allowed us to obtain a single, 7-gene plasmid in a short time using regular restriction cloning strategies. Additionally, expression of the construct in High Five™ insect cells paired with a simple 5-step purification protocol allowed the extraction of a pure, active TFIIH Core sub-complex in milligram quantities.

Subunit architecture of general transcription factor TFIIH.

Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain.

Improved methods for expression and purification of Saccharomyces cerevisiae TFIIF and TFIIH; identification of a functional Escherichia coli promoter and internal translation initiation within the N-terminal coding region of the TFIIF TFG1 subunit.

The basal RNA polymerase II (RNAPII) transcription machinery is composed of RNAPII and the general transcription factors (TF) TATA binding protein (TBP), TFIIB, TFIIE, TFIIF and TFIIH. Due to the powerful genetic and molecular approaches that can be utilized, the budding yeast Saccharomyces cerevisiae has proven to be an invaluable model system for studies of the mechanisms of RNAPII transcription. Complementary biochemical studies of the S. cerevisiae basal transcription machinery, however, have been hampered by difficulties in the purification of TFIIF and TFIIH, most notably due to the severe toxicity of the TFIIF Tfg1 subunit in Escherichia coli and the complexity of the purification scheme for native TFIIH. Here, we report the elimination of TFG1-associated toxicity in E. coli, the identification and removal of a functional E. coli promoter and internal translation initiation within the N-terminal coding region of TFG1, and the efficient production and two-step purification of recombinant TFIIF complexes. We also report conditions for the efficient two-step tandem affinity purification (TAP) of holo-TFIIH, core TFIIH and TFIIK complexes from yeast whole cell extracts.

Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems.

Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been studied in vitro using cell extracts or highly purified repair factors and radiolabeled DNA of known sequence with DNA damage at a defined site. This chapter describes procedures for preparation of DNA substrates designed for analysis of damage recognition, either the 5' or the 3' incision event, excision (resulting from concerted dual incisions), and repair synthesis. Excision in Escherichia coli is accomplished by the three-subunit Uvr(A)BC excision nuclease and in humans by six repair factors: XPA, RPA, XPChR23B, TFIIH, XPFERCC1, and XPG. This chapter outlines methods for expression and purification of these essential repair factors and provides protocols for performing each of the in vitro repair assays with either the E. coli or the human excision nuclease.

TFIIH enzymatic activities in transcription and nucleotide excision repair.

Transcription and nucleotide excision repair (NER) are two major mechanisms in which the transcription factor TFIIH plays a crucial role. In order to investigate its function, we first described a fast and efficient purification protocol of TFIIH from either HeLa cells or patient cell lines, as well as various in vitro enzymatic assays set up in our laboratory. All these enzymatic assays have been adapted to work on immobilized DNA, a powerful tool allowing for sequential protein incubations in various buffer conditions, without destabilizing protein complexes bound to the DNA. Runoff transcription assays performed with either whole cell extract or highly purified factors underline the role of TFIIH helicases (XPB and XPD) in the RNA synthesis. Moreover, the requirement of XPB and XPD in NER can also be investigated with various assays corresponding to the different steps of this process. The DNA opening assay (permanganate footprint) highlights DNA unwinding of the double-stranded DNA fragment within the repair complex, whereas the dual incision assay allows for detection of the double cut on both sides of the lesion. The gap-filling reaction following the cuts can be monitored as well with a DNA resynthesis assay. Futhermore, the use of immobilized DNA is of great interest to study the detailed mechanism in which TFIIH plays a central role. This chapter describes the ATP-independent recruitment of TFIIH on the damaged DNA previously recognized by XPC-hHR23B and the sequential arrival and departure of the repair proteins within the NER complex.