This proposal is aimed at learning the roles of the XPB and XPD subunits of the multi-protein factor TFIIH. TFIIH is essential for transcription of RNA, repair of DNA and control of cellular mitosis. XPB is essential to both transcription and repair and XPD is used for repair and mitotic control. 3 human diseases track to mutations in the XPB and XPD subunits, XP (xeroderma pigmentosum), CS (Cockaynes syndrome) and TTD (Trichothiodystrophy). TTD and CS are caused in significant part by transcription defects. There are major gaps in our knowledge of how these proteins function and how the mutated proteins contribute to CS and TTD. This proposal aims to fill those gaps. One goal is to determine how XPB leads to DNA opening during transcription. Binding and helicase assays using purified human components will be used to see how XPB and TFIIH cooperate with both basal transcription factors and activators to trigger appropriate promoter opening and transcription. In addition, mutations will be introduced into XPB in both human and fission yeast, and the effect on transcription and DNA repair determined within crude cellular extracts. Some of these mutations will mimic alleles that cause XPB diseases of transcription. A similar but less extensive analysis will be done with mutant XPD alleles. The role of other XP factors in facilitating communication between the transcription and DNA repair pathways will also be explored. These experiments are directed towards understanding whether there is an active mechanism for distributing TFIIH between transcription and DNA repair complexes. They will evaluate the roles of DNA repair factors known to bind transcription components by adding them to transcription extracts. How they interact with isolated transcription factors will also be explores to learn the extent of active communication between these 2 critical cellular processes.
| Tran, Khiem; Gralla, Jay D (2010) The TFIIB tip domain couples transcription initiation to events involved in RNA processing. J Biol Chem 285:39580-7 |
| Tran, Khiem; Gralla, Jay D (2008) Control of the timing of promoter escape and RNA catalysis by the transcription factor IIb fingertip. J Biol Chem 283:15665-71 |
| Wolner, B S; Gralla, J D (1997) Promoter activation via a cyclic AMP response element in vitro. J Biol Chem 272:32301-7 |
| Chang, C; Gralla, J D (1994) A critical role for chromatin in mounting a synergistic transcriptional response to GAL4-VP16. Mol Cell Biol 14:5175-81 |
| Jiang, Y; Gralla, J D (1994) RNA polymerase II phosphorylation: uncoupling from GAL4-VP16 directed open complex formation and transcription in a reconstituted system. Nucleic Acids Res 22:4958-62 |
| Jiang, Y; Triezenberg, S J; Gralla, J D (1994) Defective transcriptional activation by diverse VP16 mutants associated with a common inability to form open promoter complexes. J Biol Chem 269:5505-8 |
