Transcription Initiation Complex Research Articles

Transcription initiation complex structures elucidate DNA opening.

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Impeding the formation of Transcription Initiation complex

RNA synthesis is the result of a cascade of chronologically orchestrated events that requires several hundreds of proteins including RNA polymerase II, the general transcription factors, mediator, co‐activators, co‐repressors and chromatin remodelers are assembled around the promoter. Among these proteins necessary to initiate RNA synthesis, we recently identified the Nucleotide‐Excision‐Repair (NER) factors demonstrating their dual role in transcription and DNA repair. Mediator occupies a key role in protein coding genes expression in mediating the contacts between gene specific factors and the basal transcription machinery.Mutations in one of these factors results in rare genetic disorders: Xeroderma Pigmentosum (XP), Trichothiodystrophy (TTD), Cockayne Syndrome (CS), for TFIIH and NER factors; non‐syndromic mental retardation, Charcot‐Marie Tooth, Opitz‐Kaveggia … for Mediator.We show how each of the mutations in either NER factors or Mediator subunits specifically disturbs the protein/protein interactions network required for the accurate formation of the transcription complex at the promoter of activated genes.The NER proteins seem to be necessary at the promoter of activated genes in order to achieve an optimal 3D loop structure that requires DNA demethylation, formation of DNA cuts as well as histone posttranslational modifications. We also show how the Mediator mutations cause distinct expression of Immediate Early Genes (IEG) with as a consequence disruption of later genes expression.Our study shed light on the molecular mechanism underlying gene expression regulation at the transcription initiation level explaining at least partially some of the clinical features of the above‐mentioned genetic disorders.

Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3'-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E coli transcription initiation complexes (TICs) containing the stress-responsive σ(S) factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ(S)-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ(S) factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σ(S)-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ(S)-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

Genomic Organization of Human Transcription Initiation Complexes.

A repertoire of transcription initiation factors engage the core promoter of mRNA genes to recruit RNA polymerase (Pol) II to initiate transcription, yet their precise spatial organization remains unclear. Using ChIP-exo, here we detail the interactions and genomic organization of initiation factors TBP, TFIIB, and Pol II at mRNA genes and within CpG islands. We find that when Pol II moves into a transcriptionally paused state, TBP/TFIIB remain at the promoter. We show that TBP and TFIIB bound to the core promoter at two separate, resolvable locations that coincided with sites of divergent transcription initiation. We also examine the precise binding of TBP at Pol III transcribed tRNA genes. We find that TBP crosslinked to tRNA genes in a similar manner as at Pol II transcribed genes. This comprehensive and high resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events humans at Pol II coding and Pol III transcribed tRNA genes.

Suppression of mitochondrial transcription initiation complexes changes the balance of replication intermediates of mitochondrial DNA and reduces 7S DNA in cultured human cells.

Analysis of replicating mammalian mitochondrial DNA (mtDNA) suggested that initiation of the replication occurs not only at the specific position, Ori-H but also across a broad zone in mtDNA. We investigated relationship of mitochondrial transcription initiation which takes place upstream of Ori-H and mtDNA replication initiation through analysing the effect of knockdown of mitochondrial transcription factor B2, TFB2M and mitochondrial RNA polymerase, POLRMT, components of the transcription initiation complexes in cultured human cells. Under the conditions where suppression of the transcription initiation complexes was achieved by simultaneous depletion of TFB2M and POLRMT, decrease of replication intermediates of mtDNA RITOLS replication mode accompanied reduction in mtDNA copy number. On the other hand, replication intermediates of coupled leading and lagging strand DNA replication, another proposed replication mode, appeared to be less affected. The findings support the view that the former mode involves transcription from the light strand promoter (LSP), and suggest that initiation of the latter mode is independent from the transcription and has distinct regulation. Further, knockdown of TFB2M alone caused significant decrease of 7S DNA, which implies that transcription initiation complexes formed at the LSP engage 7S DNA synthesis more frequently than the initiation of productive replication and transcription.

From indole to pyrrole, furan, thiophene and pyridine: Search for novel small molecule inhibitors of bacterial transcription initiation complex formation

The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N′-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase–σ70/σA interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure–activity relationship (SAR) studies.

CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.

A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

The distal upstream promoter in Ly49 genes, Pro1, is active in mature NK cells and T cells, does not require TATA boxes, and displays enhancer activity.

Missing self recognition of MHC class I molecules is mediated in murine species primarily through the stochastic expression of CD94/NKG2 and Ly49 receptors on NK cells. Previous studies have suggested that the stochastic expression of Ly49 receptors is achieved through the use of an alternate upstream promoter, designated Pro1, that is active only in immature NK cells and operates via the mutually exclusive binding of transcription initiation complexes to closely opposed forward and reverse TATA boxes, with forward transcription being transiently required to activate the downstream promoters, Pro2/Pro3, that are subsequently responsible for transcription in mature NK cells. In this study, we report that Pro1 transcripts are not restricted to immature NK cells but are also found in mature NK cells and T cells, and that Pro1 fragments display strong promoter activity in mature NK cell and T cell lines as well as in immature NK cells. However, the strength of promoter activity in vitro does not correlate well with Ly49 expression in vivo and forward promoter activity is generally weak or undetectable, suggesting that components outside of Pro1 are required for efficient forward transcription. Indeed, conserved sequences immediately upstream and downstream of the core Pro1 region were found to inhibit or enhance promoter activity. Most surprisingly, promoter activity does not require either the forward or reverse TATA boxes, but is instead dependent on residues in the largely invariant central region of Pro1. Importantly, Pro1 displays strong enhancer activity, suggesting that this may be its principal function in vivo.

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Gene expression is highly regulated at the step of transcription initiation, and transcription activators play a critical role in this process. RbpA, an actinobacterial transcription activator that is essential in Mycobacterium tuberculosis (Mtb), binds selectively to group 1 and certain group 2 σ-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA σ-interacting domain (SID) and basic linker are sufficient for transcription activation. We also present the crystal structure of the Mtb RbpA-SID in complex with domain 2 of the housekeeping σ-factor, σ(A). The structure explains the basis of σ-selectivity by RbpA, showing that RbpA interacts with conserved regions of σ(A) as well as the nonconserved region (NCR), which is present only in housekeeping σ-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a σ-factor. We confirm the basis of selectivity and the observed interactions using mutagenesis and functional studies. In addition, the structure allows for a model of the RbpA-SID in the context of a transcription initiation complex. Unexpectedly, the structural modeling suggests that RbpA contacts the promoter DNA, and we present in vivo and in vitro studies supporting this finding. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.

Receptores a estrógenos y progesterona

La estrogenoterapia está en aumento y ello obliga a un adecuado conocimiento del mecanismo de acción de los esteroides, siendo al momento conocidos su transporte y metabolismo. Por ello discutiremos los aspectos moleculares de la acción de los estrógenos a nivel de los receptores nucleares, precisando la estructura y funcionamiento de los mismos. Se revisa los factores de transcripción, describiendo sus dominios de unión al DNA y de activación. Se hace precisiones de la transcripción, el papel de la polimerasa II y los factores generales de transcripción, que forman el complejo de iniciación de transcripción. Se culmina exponiendo cómo es la regulación de la concentración del receptor a través del mecanismo de relleno y puntualizando algunas implicancias clínicas. La estrogenoterapia está en aumento y es conveniente que su uso sea consecuencia de un adecuado conocimiento del mecanismo de acción del estrógeno en su célula blanco; este conocimiento biomolecular permite comprender la respuesta individual o grupal a esa terapia. Los receptores hormonales a estrógenos y progesterona han sido encontrados y estudiados en todo el organismo1-3 y son parte de la regulación del med¡o interno; su comprensión explica la fisiología de hormonas esteroides.

Crystal Structures of the E. coli Transcription Initiation Complexes with a Complete Bubble

During transcription initiation, RNA polymerase binds to promoter DNA to form an initiation complex containing a DNA bubble and enters into abortive cycles of RNA synthesis before escaping the promoter to transit into the elongation phase for processive RNA synthesis. Here we present the crystal structures of E. coli transcription initiation complexes containing a complete transcription bubble and de novo synthesized RNA oligonucleotides at about 6-Å resolution. The structures show how RNA polymerase recognizes DNA promoters that contain spacers of different lengths and reveal a bridging interaction between the 5'-triphosphate of the nascent RNA and the σ factor that may function to stabilize the short RNA-DNA hybrids during the early stage of transcription initiation. The conformation of the RNA oligonucleotides and the paths of the DNA strands in the complete initiation complexes provide insights into the mechanism that controls both the abortive and productive RNA synthesis.

Synthesis and biological activity of novel mono-indole and mono-benzofuran inhibitors of bacterial transcription initiation complex formation

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein–protein interaction between RNA polymerase core and σ70/σA factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase–σ70/σA interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure–activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic–hydrophobic balance is an important determinant of biological activity.

Yeast Ssu72 Protein as a Mediator of Gene Loop Formation at Bidirectional Promoters

Research over the past ten years has revealed that bidirectional promoters are a common feature of eukaryotes from yeast to metazoans. Gene loops are three-dimensional structures that inhibit divergent transcription at bidirectional promoters. Ssu72, a multifunctional protein phosphatase, has been identified as a key component in directing gene loop formation. Molecular genetic characterization first identified Ssu72 as a component of the transcription initiation complex through an interaction with TFIIB. More recently, Ssu72 has been shown to inhibit divergent transcription by simultaneously binding to both promoter and terminator elements. Here, we describe the path of Ssu72 research from its discovery as a transcriptional regulatory protein to its role in the maintenance of transcriptional memory and repression of noncoding RNAs (ncRNAs). Finally, we consider the role of Ssu72 and gene loops in human health.

Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS.

The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

Use of RNA polymerase molecular beacon assay to measure RNA polymerase interactions with model promoter fragments.

RNA polymerase-promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase-promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase-promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase-promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.

Mapping the Escherichia coli transcription elongation complex with exonuclease III.

RNA polymerase interactions with the nucleic acids control every step of the transcription cycle. These contacts mediate RNA polymerase recruitment to promoters, induce pausing during RNA chain synthesis, and control transcription termination. These interactions are dissected using footprinting assays, in which a bound protein protects nucleic acids from the digestion by nucleases or modification by chemical probes. Exonuclease III is frequently employed to study protein-DNA interactions owing to relatively simple procedures and low background. Exonuclease III has been used to determine RNA polymerase position in transcription initiation and elongation complexes and to infer the translocation register of the enzyme. In this chapter, we describe probing the location and the conformation of transcription elongation complexes formed by walking of the RNA polymerase along an immobilized template.

The Epstein-Barr virus BamHI C promoter is not essential for B cell immortalization in vitro, but it greatly enhances B cell growth transformation.

Epstein-Barr virus (EBV) infection of B cells leads to the sequential activation of two viral promoters, Wp and Cp, resulting in the expression of six EBV nuclear antigens (EBNAs) and the viral Bcl2 homologue BHRF1. The viral transactivator EBNA2 is required for this switch from Wp to Cp usage during the initial stages of infection. EBNA2-dependent Cp transcription is mediated by the EBNA2 response element (E2RE), a region that contains at least two binding sites for cellular factors; one of these sites, CBF1, interacts with RBP-JK, which then recruits EBNA2 to the transcription initiation complex. Here we demonstrate that the B cell-specific transcription factor BSAP/Pax5 binds to a second site, CBF2, in the E2RE. Deletion of the E2RE in the context of a recombinant virus greatly diminished levels of Cp-initiated transcripts during the initial stages of infection but did not affect the levels of Wp-initiated transcripts or EBNA mRNAs. Consistent with this finding, viruses deleted for the E2RE were not markedly impaired in their ability to induce B cell transformation in vitro. In contrast, a larger deletion of the entire Cp region did reduce EBNA mRNA levels early after infection and subsequently almost completely ablated lymphoblastoid cell line (LCL) outgrowth. Notably, however, rare LCLs could be established following infection with Cp-deleted viruses, and these were indistinguishable from wild-type-derived LCLs in terms of steady-state EBV gene transcription. These data indicate that, unlike Wp, Cp is dispensable for the virus' growth-transforming activity. Epstein-Barr virus (EBV), a B lymphotropic herpesvirus etiologically linked to several B cell malignancies, efficiently induces B cell proliferation leading to the outgrowth of lymphoblastoid cell lines (LCLs). The initial stages of this growth-transforming infection are characterized by the sequential activation of two viral promoters, Wp and Cp, both of which appear to be preferentially active in target B cells. In this work, we have investigated the importance of Cp activity in initiating B cell proliferation and maintaining LCL growth. Using recombinant viruses, we demonstrate that while Cp is not essential for LCL outgrowth in vitro, it enhances transformation efficiency by >100-fold. We also show that Cp, like Wp, interacts with the B cell-specific activator protein BSAP/Pax5. We suggest that EBV has evolved this two-promoter system to ensure efficient colonization of the host B cell system in vivo.

Transcription Regulation Targeting in Peripheral T Cell Lymphomas Induces Apoptosis and Sensitization to BCL2 Inhibitors

Peripheral T-cell lymphomas (PTCL) are clinically aggressive diseases with poor response to available (largely B-cell lymphoma–tailored) chemotherapy regimens and dismal survival.To identify active drugs for PTCL patients, we performed a cell-based progressive screen from a library of 105 anti-neoplastic drugs in clinical use. Primary screening was done in the PTCL-NOS cell line (OCI-Ly12), using three drug concentrations. We identified 3 active drug groups within the clinical-range limit: HDAC inhibitors (HDI) (romidepsin), proteasome inhibitors (bortezomib, carfilzomib) and transcription inhibitors (dactinomycin). Secondary screening was conducted for the active drug groups with 6 drug concentrations, in an extended panel of 9 TCL cell lines. We also expanded the drugs in each group by adding vorinostat, panobinostat and valproic acid for HDI and SNS032 (CDK9>2>>7 inhibitor) and THZ1 (CDK7>12 inhibitor) for transcription inhibitors. We demonstrated that the most active drugs (within serum achievable concentrations) were bortezomib, carfilzomib, romidepsin, dactinomycin and THZ1. Since proteasome inhibitors and romidepsin are being currently tested in PTCL clinical trials, we focused on the transcriptional inhibitors. Actinomycin inhibits the transcription initiation complex and prevents RNA elongation by RNA-POL2; given this broad mechanism of action, it is associated with serious side effects that limit its clinical use either alone or in combination. On the other hand, a recently discovered CDK7>12 inhibitor, THZ1, showed minimal side effects in vivo (Kwiatkowski N, et al. Nature 2014 Jun22), and high potency in our secondary screening yielding IC50s in TCL of ~200nM. We therefore used THZ1 to investigate the functional relevance of CDK7/12 targeting in PTCL. CDK7 is a critical component of both CDK-activating kinase (CAK) and transcription factor II human (TFIIH) complexes that phosphorylates cell cycle CDKs and C-terminal domain (CTD) of RNA-POL2, respectively. CDK7 inhibition decreases the phosphorylation of RNA-POL2 at Ser5 and Ser2, leading to transcriptional inhibition of susceptible loci. CDK12 in complex with CCNK displays a CTD kinase activity that is required for RNA splicing. To better understand the sensitivity of TCL to THZ1, we first determined the expression of all CAK components (CDK7, CCNH, and MAT1A) and CDK12/CCNK in 9 TCL cell lines that were found overexpressed compared to normal T cells from tonsils. Treatment of OCI-Ly12 and OCI-Ly13.2 (ALCL-ALK negative) cell lines with THZ1 for up to 48 h induced dose- and time-dependent decrease of phospho-POL2-ser5 and phospho-POL2-ser2, followed by PARP cleavage, caspase 7/3 activation and apoptosis. Genes with super-enhancers were found to be more susceptible to THZ1, and we also found these enhancers in genes associated with PTCL prognosis such as MCL1, JAK1 and MYC. Accordingly, THZ1 decreased mRNA and protein levels of MCL1, JAK1 and MYC as early as 3 hours after treatment. This was followed by decreasing levels of BCL2, BCL-XL, JUND and NFkB, and increased expression of pro-apoptotic proteins such as BAX at later time points. The decrease in JAK1 abundance led up to 70% reduction of phospho-STAT3 activity (as determined by immunoblotting and EMSA-like assays). Moreover, induction of STAT3 phosphorylation by IL-7/IL-15 treatment partially rescued the effect of THZ1, suggesting that JAK1 is a relevant target of CDK7 in PTCLs. The THZ1-dependent decrease in anti-apoptotic BCL2-family members prompted us to determine whether CDK7/12 inhibition can re-sensitize TCL to these drugs. We found either synergistic or re-sensitization effects on combination of THZ1 with BH3-family inhibitors, ABT-737 and obatoclax, in both OCI-LY12 and OCI-Ly13.2 cells.In sum, we identified a mechanism by which CDK7/12 inhibition, with the irreversible clinical candidate compound THZ1, simultaneously inhibits prominent PTCL survival pathways (JAK/STAT3, MYC and BCL2) causing apoptosis and re-sensitization to BCL2-family inhibitors. DisclosuresOff Label Use: THZ1 (CDK7 and CDK12 inhibitor) for inducing apoptosis and sensitization to BCL2 inhibitors in peripheral T cell lymphomas.. Kwiatkowski:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Zhang:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Gray:Syros Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Coupling of Downstream RNA Polymerase–Promoter Interactions with Formation of Catalytically Competent Transcription Initiation Complex

Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP–promoter interactions upstream of the transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP–downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.

E(y)1/TAF9 mediates the transcriptional output of Notch signaling in Drosophila

Transcriptional activation of Notch signaling targets requires the formation of a ternary complex that involves the intracellular domain of the Notch receptor (NICD), DNA-binding protein Suppressor of Hairless [Su(H), RPBJ in mammals] and coactivator Mastermind (Mam). Here, we report that E(y)1/TAF9, a component of the transcription factor TFIID complex, interacts specifically with the NICD-Su(H)-Mam complex to facilitate the transcriptional output of Notch signaling. We identified E(y)1/TAF9 in a large-scale in vivo RNA interference (RNAi) screen for genes that are involved in a Notch-dependent mitotic-to-endocycle transition in Drosophila follicle cells. Knockdown of e(y)1/TAF9 displayed Notch-mutant-like phenotypes and defects in target gene and activity reporter expression in both the follicle cells and wing imaginal discs. Epistatic analyses in these two tissues indicated that E(y)1/TAF9 functions downstream of Notch cleavage. Biochemical studies in S2 cells demonstrated that E(y)1/TAF9 physically interacts with the transcriptional effectors of Notch signaling Su(H) and NICD. Taken together, our data suggest that the association of the NICD-Su(H)-Mastermind complex with E(y)1/TAF9 in response to Notch activation recruits the transcription initiation complex to induce Notch target genes, coupling Notch signaling with the transcription machinery.