Podcasts about tfiif

Gene transcription is a fundamental process of the living cell. Eukaryotic transcription of messenger RNA requires the regulated recruitment of the conserved transcribing enzyme RNA polymerase (Pol) II to the gene promoter. The most heavily regulated step is transcription initiation that involves the ordered assembly of Pol II, the general transcription factors (TF) -IIA, -IIB, -IID, -IIF, -IIE, -IIH and the co-activator Mediator complex. Mediator communicates between transcription regulators and Pol II, and is associated with human disease. Mediator from the yeast Saccharomyces cerevisiae (Sc) has a molecular mass of 1.4 megadaltons and contains 25-subunits that constitute a head, middle, tail and kinase module. The core of Mediator contains the head and middle modules that are essential for viability in Sc, and directly contact Pol II. Mediator co-operates with TFIIH, to assist assembly and stabilization of the transcription initiation complex and stimulate TFIIH kinase activity. Because of the large size and complexity of Mediator and the initiation machinery, the underlying mechanism remains poorly understood. In this work we studied the structure and function of Mediator head and middle modules, the structure of the reconstituted Pol II-core Mediator transcription initiation complex, and reveal mechanisms of transcription regulation. We report the crystal structure of the 6-subunit Schizosaccharomyces pombe Mediator head module at 3.4 Å resolution. The structure resembles the head of a crocodile and reveals eight elements that are part of three domains named neck, fixed jaw and movable jaw. The neck contains a spine, shoulder, arm and finger. The arm and essential shoulder elements contact the remainder of Mediator and Pol II. The head module jaws and central joint, important for transcription, also interact with Mediator and Pol II. The Sp head module structure is conserved and revises a 4.3 Å model of the Sc head module, explains known mutations, and provides an atomic model for one half of core Mediator. We further propose a model of the Mediator middle module based on protein crosslinking and mass spectrometry. To determine how Mediator regulates initiation, we prepared recombinant Sc core Mediator by co-expression of its 15 subunits in bacteria. Core Mediator is active in transcription assays and bound an in vitro reconstituted core initially transcribing complex (cITC) that contains Pol II, the general factors TFIIB, TBP, TFIIF, and promoter DNA. We determined the cryo-electron microscopy structure of the initially transcribing core initiation complex at 7.8 Å resolution. The structure reveals the arrangement of DNA, TBP, TFIIB, and TFIIF on the Pol II surface, the path of the complete DNA template strand and three TFIIF elements. The ‘charged helix’ and ‘arm’ of TFIIF subunit Tfg1, reach into the Pol II cleft and may stabilize open DNA. The linker region of TFIIF subunit Tfg2 extends between Pol II protrusion and TFIIB, and may stabilize TFIIB. The structure agrees with its human counterpart, and suggests a conserved architecture of the core initiation complex. Finally, we determined the cryo-electron microscopy architecture of the cITC-core Mediator complex at 9.7 Å resolution. Core Mediator binds Pol II at the Rpb4/Rbp7 stalk close to the carboxy-terminal domain (CTD). The Mediator head module contacts the Pol II dock and TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module ‘plank’ domain touches the Pol II foot and may control polymerase conformation allosterically. The Med14 subunit bridges head and middle modules with a ‘beam’, and connects to the tail module that binds transcription activators located on upstream DNA. The ‘arm’ and ‘hook’ domains of core Mediator form part of a ‘cradle’ that may position CTD and the TFIIH kinase to stimulate Pol II phosphorylation. Taken together, our results provide a structural framework to unravel the role of Mediator in transcription initiation and determine mechanisms of gene regulation.

Eukaryotic nuclear transcription is carried out by three different Polymerases (Pol), Pol I, Pol II and Pol III. Among these, Pol I is dedicated to transcription of the rRNA, which is the first step of ribosome biogenesis, and cell growth is regulated during Pol I transcription initiation by the conserved factor Rrn3/TIF-IA in yeast/human. A wealth of structural information is available on Pol II and its general transcription factors (GTFs). Recently, also the architectures of Pol I and Pol III have been described by electron microscopy and the additional subunits that are specific to Pol I and Pol III have been identified as orthologs of the Pol II transcription factors TFIIF and TFIIE. Nevertheless, we still lack information about the architecture of the Pol I initiation complex and structural data is missing explaining the regulation of Pol I initiation mediated by its central transcription initiation factor Rrn3. The Rrn3 structure solved in this study reveals a unique HEAT repeat fold and indicates dimerization of Rrn3 in solution. However, the Rrn3-dimer is disrupted upon Pol I binding. The Rrn3 structure further displays a surface serine patch. Phosphorylation of this patch represses human Pol I transcription (Mayer et al, 2005; Mayer et al, 2004), and a phospho-mimetic patch mutation prevents Rrn3 binding to Pol I in vitro, and reduces S. cerevisiae cell growth and Pol I gene occupancy in vivo. This demonstrates a conserved regulation mechanism of the Pol I-Rrn3 interaction. Crosslinking indicates that Rrn3 does not only interact with Pol I subunits A43/14, but the interface further extends past the RNA exit tunnel and dock domain to AC40/19. The corresponding region of Pol II binds the Mediator head (Soutourina et al., 2011) that co-operates with TFIIB (Baek et al, 2006). Consistent with this, the Rrn3 binding partner, core factor subunit Rrn7, is predicted to be a TFIIB homologue. Taken together, our results provide the molecular basis of Rrn3-regulated Pol I initiation and cell growth and indicate a universally conserved architecture of eukaryotic transcription initiation complexes.

RNA polymerase II (RNAPII) has been identified almost 40 years ago, but the molecular details of its regulation and fine tuning during messenger RNA (mRNA) synthesis are still far from understood. Subsequently to RNAPII six general transcription factors (GTFs; TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) were discovered of which all except TFIIA are necessary and sufficient for promoter-dependent basal transcription initiation. In addition to the GTFs activator-dependent transcription requires the presence of a transcription cofactor, the Mediator complex. Mediator serves as a link between transcription activators, enhancers and the general transcription machinery. Initial studies revealed that Mediator stimulates the activity of the TFIIH associated kinase CDK7 and thereby facilitates RNAPII C-terminal domain (CTD) phosphorylation. Furthermore the Mediator complex interacts functionally with several signal transduction pathways and serves as an signal integration platform. In order to dissect the process of transcription initiation, early studies made use of in vitro transcription systems reconstituted from recombinant or highly purified GTFs and RNAPII. In this system basal, activator-independent transcription does not require the presence of the Mediator complex. If however a more physiological nuclear extract transcription system is used, our laboratory and others have established previously that basal transcription becomes critically dependent on Mediator. Another difference between both transcription systems is that the first is insensitive to the kinase inhibitor H8 whereas in the second transcription can be inhibited by H8. This suggests that only the second transcription system is regulated by RNAPII CTD phosphorylation. In this thesis the interplay between Mediator, RNAPII, GTFs and transcription cofactors was studied using immobilized promoter template assays in combination with various immunodepleted nuclear extracts and recombinant factors. Negative cofactor 2 (NC2) is an evolutionary conserved general cofactor that binds to many active genes in vivo. Previous studies in our laboratory had shown with recombinant proteins that NC2 competes with TFIIA and TFIIB for binding to TATA-binding protein (TBP) at a promoter in vitro. Genetic studies in yeast provided evidence that Mediator acts antagonistically to NC2. Here I have studied the role of NC2 on preinitiation complex (PIC) formation and transcription in nuclear extracts. I observed rapid association of TFIID with promoters whereas NC2 enters PICs with a slow kinetic which is similar to that of TFIIB recruitment. My data indirectly suggest that TBP binds to DNA in a yet to be defined inactive form (perhaps as a TFIID complex) which is then slowly converted into an active TBP-TATA complex that is rapidly recognized by GTFs or NC2. My data support the notion that NC2 and TFIIB compete for binding to a PIC also in immobilized promoter assays under physiological conditions. NC2 concentrations in nuclear extracts appears to be tightly controlled. Doubling the NC2 concentration in a nuclear extract by adding recombinant NC2 (rNC2) abolished functional PIC formation and transcription. However, the in vitro analysis also showed that upstream of NC2 PIC formation is fully dependent on Mediator. Hence, TFIID binds to a promoter in a nuclear extract in vitro transcription system but we have no indication that a transcription competent PIC is formed in the absence of Mediator. In yeast studies it was reported that upon transcription initiation in vitro several GTFs dissociate from the promoter DNA template whereas the Mediator complex is retained in a reinitiation complex. In the human system I recapitulate this observation for TFIIB and CDK7. In addition I provide evidence that Mediator partially dissociated from the promoter template upon transcription initiation. Upon transcription initiation the middle module subunit MED7 was retained on a promoter template, whereas the tail module subunit MED15 and CDK8 did dissociate. This data suggest that upon transcription initiation a head/middle module Mediator subcomplex is retained at the promoter whereas the tail and CDK8 modules dissociate. Previous studies have established that Mediator promotes CDK7-dependent phosphorylation of the RNAPII CTD at serine-5 (ser-5). Various studies found that CTD ser-5 phosphorylation does coincide with transcription initiation. Using new monoclonal antibodies I observed two functionally distinct modes of CTD ser-5 phosphorylation in vitro: Hypo- and hyperphosphorylation of the largest RNAPII subunit Rpb1. I observed that CTD ser-5 hypophosphorylation is established already before complex opening by TFIIH. I found CTD ser-5 hypophosphorylation to be critically dependent on TBP, Mediator, TFIIB and CDK7. In addition I noted that CTD ser-5 hypophosphorylation correlates with the transcription potential of a PIC. CTD ser-5 hyperphosphorylation was established in a Mediator-dependent fashion but independent of productive transcription. Immunodepletion of CDK7 did not led to a reduction in CTD ser-5 hyperphosphorylation. However, immunodepletion of CDK8 caused a reduction but not a loss of CTD ser-5 hyperphosphorylation upon transcription initiation indicating that another yet to be identified kinase might be involved in this process. These data suggest that CTD ser-5 hypophosphorylation is established only in the PIC context on RNAPII located at bona fide promoter regions but not on RNAPII complexes bound to DNA outside of promoter regions, e.g. in an open reading frame. Recently phosphorylation of the RNAPII CTD at serine-7 (ser-7) was reported. In that study the entire coding region of the TCRβ locus was found to be associated with RNAPII CTD phosphorylated at ser-7. Starting from there I found that establishment of CTD ser-7 phosphorylation in the process of transcription initiation can be recapitulated in an immobilized template assay system in vitro. I confirmed the in vitro finding that establishment of CTD ser-7 phosphorylation correlates with transcription initiation with chromatin immunoprecipitation experiments on an inducible model gene system in vivo. Similar to CTD ser-5 phosphorylation, I observed two modes of CTD ser-7 phosphorylation: CTD ser-7 hypo- and hyperphosphorylation. In contrast to CTD ser-5 hypophosphorylation, which was established before complex opening, I observed establishment of CTD ser-7 hypophosphorylation predominantly after complex opening by TFIIH. Both, CTD ser-7 hypo- and hyperphosphorylation were found to be Mediator-dependent. A mass spectrometric screen for PIC associated kinases (in collaboration with the laboratory of Gerhard Mittler) yielded 13 kinases. Seven of the identified kinases were further tested for their potential to phosphorylate the RNAPII at ser-7 in an immobilized template assay.

Synthesis of ribosomal RNA by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. In this thesis a reproducible large-scale purification protocol for Pol I from S. cerevisiae could be developed. Crystals were obtained, diffraction to < 4 Å could be recorded, however, the enormously complex non-crystallographic symmetry impeded structure solution. Switching to cryo-electron microscopy, the structure of the complete 14-subunit enzyme could be solved to 12 Å resolution, a homology model for the core enzyme could be generated, and the crystal structure of the subcomplex A14/43 could be solved. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor, and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3’-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2, and apparently enables rRNA proofreading and 3’-end trimming.

TFIIF is the only general transcription factor that has been implicated in the preinitiation complex assembly, open complex formation, initiation and transcription elongation. In addition, TFIIF stimulates Fcp1, a central phosphatase needed for recycling of RNA polymerase II (Pol II) after transcription by dephosphorylation of the Pol II C-terminal domain (CTD). This thesis reports the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to phosphotransferases and –hydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. It was further demonstrated that the first aspartate in the signature motif undergoes metalassisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for Fcp1 binding to the polymerase in vitro. Until now, the main impediment in the high resolution crystallographic studies of TFIIF in complex with Pol II and other members of transcription machinery was unavailability of soluble, stoichiometric TFIIF complex in sufficient amounts. This thesis reports on the development of the overexpression system in yeast and a purification protocol that enabled for the first time to isolate milligram amounts of a pure and soluble, 15-subunit (~0,7 MDa) stoichiometric Pol IITFIIF complex. Such complex together with the promoter DNA, RNA, TBP and TFIIB assembles in vitro into the yeast initially transcribing complex, which can now be studied structurally.