Podcasts about tfiia

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.

Initiation of transcription by eukaryotic RNA polymerase II is finely controlled by a multitude of regulatory factors. Among them, the negative cofactor 2 (NC2), composed of the subunits NC2alpha and NC2beta, is able to bind directly to TBP-DNA complexes, preventing the assembly of the general transcription factors TFIIA and TFIIB. Despite extensive research on the negative and positive function of NC2, several questions concerning its regulation remain unexplored. In particular, localization and post-translational modifications are poorly understood. This work is the first to give some insights on the regulation of this factor. We present evidence that both subunits contain a nuclear localization signal (NLS) responsible for the accumulation of proteins in the nucleus. Immunofluorescence studies showed that NC2 dimer localizes exclusively in the nucleoplasm. However, the two subunits reveal characteristic and unique distribution patterns: NC2alpha is also found in the nucleoli, and NC2beta in small concentrations also in the cytoplasm. Moreover, we show that the two subunits already dimerize in the cytoplasm and are transported into the nucleus as a complex. Interestingly, both NLS are essential for import of the dimer. We also report for the first time several isoforms of both subunits. In vivo labeling experiments showed that NC2alpha is specifically hyperphosphorylated during mitosis. This modification does not impair its ability to dimerize with the partner and bind to TBP-DNA complexes, nor affects the stability of the complex. Furthermore, the phosphorylated protein maintains the ability to mobilize TBP on the DNA. These results suggest that NC2 is still bound to DNA during mitosis, in line with the idea that this factor keeps TBP stably associated to DNA.

Biochemische Untersuchungen beschreiben NC2 als einen Transkriptionsrepressor, welcher stabil an das TATA-Box bindende Protein (TBP) bindet. Die spezifische Bindung von NC2 an TBP inhibiert die weitere Anlagerung der generellen Transkriptionsfaktoren TFIIA und TFIIB und führt dadurch zur Unterbrechung der Bildung des Initiationskomplexes. NC2 besteht aus zwei Untereinheiten, NC2a und NC2b, die starke Homologien zu den Histonen H2A bzw. H2B aufweisen. Alle Erkenntnisse zu Beginn dieser Arbeit basierten auf Beobachtungen, die in vitro erhalten wurden. Unklar sind die Funktionen von NC2 in der Zelle. Die Aufgabe dieser Arbeit bestand darin, ein in vivo-Modellsystem zu etablieren. Als Modellorganismus wurde die Bäckerhefe Saccharomyces cerevisiae ausgewählt. Hefe hat zwei Proteine, die stark homolog zum menschlichen NC2 sind. Beide sind essentiell für das vegetative Wachstum von Hefe. Für Plasmid-Austausch-Experimente wurden Hefestämme konstruiert, bei denen die chromosomalen Gene für NC2a und NC2b durch Wildtyp-Kopien auf einem URA3-Plasmid ersetzt wurden. Mit Hilfe einer negativen Selektion gegen das URA3-Gen in Anwesenheit von 5-FOA gelang es, die humanen NC2a- und NC2b-Gene als episomale Kopien in Hefe stabil einzubringen. So zeigte sich unter anderem, daß die beiden humanen NC2-Untereinheiten, sowohl einzeln in Kombination mit ihrem Dimerisierungspartner aus Hefe als auch gemeinsam in Form des menschlichen binären Komplexes, fähig waren, die physiologische Funktion ihres Gegenstückes aus Hefe zu übernehmen. Das gleiche System wurde auch eingesetzt, um Deletionsmutanten der humanen NC2-Gene in vivo zu untersuchen. Es wurde festgestellt, daß in beiden NC2-Untereinheiten die Domänen, welche für die in vivo-Funktion notwendig sind, die vom Mensch zur Hefe konservierten Regionen enthalten. Ein wesentlicher Teil dieser Arbeit bestand darin, spontane Suppressoren einer limitierenden NC2-Funktion in vivo zu isolieren und Suppressoren mit genomischen Punktmutationen zu charakterisieren. Gefunden wurde eine Punktmutation in der großen Untereinheit (Toa1) des Hefe-TFIIA, welche einen einzigen Aminosäure-Austausch von Valin zu Phenylalanin verursacht. Hefezellen, die diese Suppressor-Mutation in Toa1 (mt- Toa1) tragen, weisen einen Kälte-sensitiven Phänotyp auf und sind trotz fehlender NC2-Gene lebensfähig. Die biochemischen Eigenschaften des rekombinanten Proteins der Suppressor-Mutante wurden durch Gelretardations-, Footprinting- und in vitro Transkriptionsexperimente untersucht. Das Protein mt-Toa1 war in der Lage, stabile TFIIA-Komplexe zusammen mit der kleinen Untereinheit Toa2 auszubilden und die Rekrutierung von TBP an die TATA-Box auf dem Promotor zu unterstützen. Allerdings zeigten weitere Untersuchungen der Suppressor-Mutante, daß der ternäre Komplex aus mt-yTFIIA, TBP und DNA weniger stabil ist. Hinweise darauf gab die reduzierte Menge an Protein-DNA-Komplexen im Fall von mtyTFIIA in Gelretardationsexperimenten unter sättigenden Bedingungen. Das mt-yTFIIA verlor zugleich seine Antirepressionsaktivität in in vivo-Transkriptionsexperimenten in Anwesenheit von NC2. Die Isolierung und Charakterisierung der Suppressor-Mutante von NC2 lieferten zum ersten Mal den Beweis, daß die Genregulation in vivo eine präzise Balance zwischen positiv und negativ wirkenden Aktivitäten erfordert. Gleichzeitig bestätigen sie die in vitro Beobachtungen, insbesondere das Gleichgewicht zwischen TFIIA und NC2 in der Kompetition um das TATA-bindende Protein TBP. Eine weitere Aufgabe der Arbeit waren Mutagenese-Studien von humanem NC2 in vivo und in vitro. Innerhalb des Histone-Fold-Motives beider NC2-Untereinheiten wurden Punktmutanten isoliert, die ihre essentielle Funktion in der Hefezelle vollständig verloren haben. Es konnten Mutanten identifiziert werden, die Einfluß auf das Wachstum der Hefe mit dem Verlust der in vitro-Aktivität korrelierten. Die Charakterisierung dieser Mutanten lieferte erste Hinweise auf funktionelle Oberflächen von NC2, die für die ternäre Komplexbildung (NC2-TBP-DNA) und die Repressionsfunktion wichtig sind. Zusammengefaßt schafft die vorliegende Arbeit einen Einblick in die NC2-Funktion in der Zelle und erweitert unser Verständnis über den molekularen Mechanismus der Transkriptionsregulation während der Initiation der Klasse II-Transkription.