Tfb1 Subunit Research Articles

Structural polymorphism of the PH domain in TFIIH.

The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.

Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER

XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4–Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4–Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription.

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3′-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2–Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.

NMR Structure of the Complex between the Tfb1 Subunit of TFIIH and the Activation Domain of VP16: Structural Similarities between VP16 and p53

The Herpes Simplex Virion Protein 16 (VP16) activates transcription through a series of protein/protein interactions involving its highly acidic transactivation domain (TAD). The acidic TAD of VP16 (VP16TAD) has been shown to interact with several partner proteins both in vitro and in vivo, and many of these VP16 partners also bind the acidic TAD of the mammalian tumor suppressor protein p53. For example, the TADs of VP16 and p53 (p53TAD) both interact directly with the p62/Tfb1 (human/yeast) subunit of TFIIH, and this interaction correlates with their ability to activate both the initiation and elongation phase of transcription. In this manuscript, we use NMR spectroscopy, isothermal titration calorimetery (ITC) and site-directed mutagenesis studies to characterize the interaction between the VP16TAD and Tfb1. We identify a region within the carboxyl-terminal subdomain of the VP16TAD (VP16C) that has sequence similarity with p53TAD2 and binds Tfb1 with nanomolar affinity. We determine an NMR structure of a Tfb1/VP16C complex, which represents the first high-resolution structure of the VP16TAD in complex with a target protein. The structure demonstrates that like p53TAD2, VP16C forms a 9-residue alpha-helix in complex with Tfb1. Comparison of the VP16/Tfb1and p53/Tfb1 structures clearly demonstrates how the viral activator VP16C and p53TAD2 shares numerous aspects of binding to Tfb1. Despite the similarities, important differences are observed between the p53TAD2/Tfb1 and VP16C/Tfb1 complexes, and these differences demonstrate how selected activators such as p53 depend on phosphorylation events to selectively regulate transcription.

NMR Structure of the Amino-Terminal Domain from the Tfb1 Subunit of TFIIH and Characterization of Its Phosphoinositide and VP16 Binding Sites,

General transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIEalpha. TFIIH has also been shown to interact with a number of transcriptional activator proteins through interactions with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal domain from the Tfb1 subunit of yeast TFIIH (Tfb1(1-115)). Like the corresponding domain from the human p62 protein, Tfb1(1-115) contains a PH domain fold despite a low level of sequence identity between the two functionally homologous proteins. In addition, we have performed in vitro binding studies that demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides [PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site on Tfb1 (p62) is located in the basic pocket formed by beta-strands beta5-beta7 of the PH domain fold. Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation.

Letter to the Editor: 1H, 15N, and 13C resonance assignment of the amino-terminal domain of the Tfb1 subunit of yeast TFIIH

Letter to the Editor: 1H, 15N, and 13C resonance assignment of the amino-terminal domain of the Tfb1 subunit of yeast TFIIH

Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH).

The Rad2, Rad3, Rad4, and Ss12 proteins are required for nucleotide excision repair in yeast cells and are homologs of four human proteins which are involved in a group of hereditary repair-defective diseases. We have previously shown that Rad3 protein is one of the five subunits of purified RNA polymerase II basal transcription initiation factor b (TFIIH) and that Ss12 protein physically associates with factor b (W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg, Cell 75:1379-1387, 1993). Here we show that the Rad2 and Rad4 proteins interact with purified factor b in vitro. Rad2 (a single-stranded DNA endonuclease) specifically interacts with the Tfb1 subunit of factor b, and we have mapped a limited region of the Rad2 polypeptide which is sufficient for this interaction. Rad2 also interacts directly with Ss12 protein (a putative DNA helicase). The binding of Rad2 and Rad4 proteins to factor b may define intermediates in the assembly of the nucleotide excision repair repairosome. Furthermore, the loading of factor b (or such intermediates) onto promoters during transcription initiation provides a mechanism for the preferential targeting of repair proteins to actively transcribing genes.

Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH).

The Rad2, Rad3, Rad4, and Ss12 proteins are required for nucleotide excision repair in yeast cells and are homologs of four human proteins which are involved in a group of hereditary repair-defective diseases. We have previously shown that Rad3 protein is one of the five subunits of purified RNA polymerase II basal transcription initiation factor b (TFIIH) and that Ss12 protein physically associates with factor b (W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg, Cell 75:1379-1387, 1993). Here we show that the Rad2 and Rad4 proteins interact with purified factor b in vitro. Rad2 (a single-stranded DNA endonuclease) specifically interacts with the Tfb1 subunit of factor b, and we have mapped a limited region of the Rad2 polypeptide which is sufficient for this interaction. Rad2 also interacts directly with Ss12 protein (a putative DNA helicase). The binding of Rad2 and Rad4 proteins to factor b may define intermediates in the assembly of the nucleotide excision repair repairosome. Furthermore, the loading of factor b (or such intermediates) onto promoters during transcription initiation provides a mechanism for the preferential targeting of repair proteins to actively transcribing genes.