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Control of interactions among proteins is critical in the treatment of diseases, but the specificity required is not easily incorporated into small molecules. Macromolecules could be more suitable as antagonists in this situation, and RNA aptamers have become particularly promising. Here we describe a novel selection procedure for RNA aptamers against a protein that constitutes a single structural domain, the Drosophila TATA-binding protein (TBP). In addition to the conventional filter partitioning method with free TBP as target, we performed another experiment, in which the TATA-bound form of TBP was targeted. Aptamers generated by both selections were able to bind specifically to TBP, but the two groups showed characteristics which were clearly different in terms of their capability to compete with TATA-DNA, their effects on the TATA-bound form of TBP, and their effects on in vitro transcription. The method used to generate these two groups of aptamers can be used with other targets to direct aptamer specificity to discrete sites on the surface of a protein.

Transcriptional regulation in retroviruses resides in the U3 region of the proviral long terminal repeat (LTR). Transcription binding sites (TBS) in the U3 region of proviral sequences derived from the milk of 17 goats infected with caprine arthritis–encephalitis virus (CAEV) were analysed by nested PCR and sequencing. U3 sequences shared a high degree of homology (86–99%) and were closely related to isolates previously ascribed to small ruminant lentivirus subtype B1. Multiple putative AP-1, AP-4, Ets-1, Stat-1 and TATA binding protein (TBP) sites were highly conserved (>85% of isolates), as were single AML(vis), GAS, IRF-1, NFAT and TAS sites. A 10 nucleotide insertion of undetermined relevance was identified in the U3 region of two isolates. To study the stability of TBS within the CAEV U3 region through in vitro passage, milk-derived isolates of CAEV from three infected dams were cultured in goat synovial membrane (GSM) cells; in one isolate the viral U3 region was completely stable during in vitro passage, in a second isolate the viral U3 region accumulated multiple deletions, single nucleotide polymorphisms and insertions, while a third isolate had an intermediate degree of promoter stability. Promoter mutations arising during in vitro passage did not affect most of the conserved putative TBS identified in CAEV.

The TATA binding protein (TBP) is an essential transcription initiation factor in Archaea and Eucarya. Bacteria lack TBP, and instead use sigma factors for transcription initiation. TBP has a symmetric structure comprising two repeated TBP domains. Using sequence, structural and phylogenetic analyses, we examine the distribution and evolutionary history of the TBP domain, a member of the helix-grip fold family. Our analyses reveal a broader distribution than for TBP, with TBP-domains being present across all three domains of life. In contrast to TBP, all other characterized examples of the TBP domain are present as single copies, primarily within multidomain proteins. The presence of the TBP domain in the ubiquitous DNA glycosylases suggests that this fold traces back to the ancestor of all three domains of life. The TBP domain is also found in RNase HIII, and phylogenetic analyses show that RNase HIII has evolved from bacterial RNase HII via TBP-domain fusion. Finally, our comparative genomic screens confirm and extend earlier reports of proteins consisting of a single TBP domain among some Archaea. These monopartite TBP-domain proteins suggest that this domain is functional in its own right, and that the TBP domain could have first evolved as an independent protein, which was later recruited in different contexts.

We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders.

Transcriptional activators coordinate the dynamic assembly of multiprotein coactivator complexes required for gene expression to occur. Here we combine the power of in vivo covalent chemical capture with p-benzoyl-L-phenylalanine (Bpa), a genetically incorporated photo-crosslinking amino acid, and chromatin immunoprecipitation (ChIP) to capture the direct protein interactions of the transcriptional activator VP16 with the general transcription factor TBP at the GAL1 promoter in live yeast.

Some variations of human genome [for example, single nucleotide polymorphisms (SNPs)] are markers of hereditary diseases and drug responses. Analysis of them can help to improve treatment. Computer-based analysis of millions of SNPs in the 1000 Genomes project makes a search for SNP markers more targeted. Here, we combined two computer-based approaches: DNA sequence analysis and keyword search in databases. In the binding sites for TATA-binding protein (TBP) in human gene promoters, we found candidate SNP markers of gender-biased autoimmune diseases, including rs1143627 [cachexia in rheumatoid arthritis (double prevalence among women)]; rs11557611 [demyelinating diseases (thrice more prevalent among young white women than among non-white individuals)]; rs17231520 and rs569033466 [both: atherosclerosis comorbid with related diseases (double prevalence among women)]; rs563763767 [Hughes syndrome-related thrombosis (lethal during pregnancy)]; rs2814778 [autoimmune diseases (excluding multiple sclerosis and rheumatoid arthritis) underlying hypergammaglobulinemia in women]; rs72661131 and rs562962093 (both: preterm delivery in pregnant diabetic women); and rs35518301, rs34166473, rs34500389, rs33981098, rs33980857, rs397509430, rs34598529, rs33931746, rs281864525, and rs63750953 (all: autoimmune diseases underlying hypergammaglobulinemia in women). Validation of these predicted candidate SNP markers using the clinical standards may advance personalized medicine.

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

In this work, we present a novel energy-transfer (ET)-based photoelectrochemical (PEC) probing of DNA–protein interactions, which associates intimately with many important intracellular processes in transcriptional regulatory networks. Specifically, Au nanoparticles (NPs) were confined onto the CdS quantum dots (QDs) functionalized PEC surface by the formation of duplex DNA, the subsequent binding of the TATA binding protein (TBP) and the resulting distortion of the Au NPs capped DNA sequence could adjust the interparticle distance and thereby modulate the PEC performance of CdS QDs through the ET process between the CdS QDs and Au NPs. Using the duplex DNA sequence as a rigid spacer, the relationship between the photocurrent quenching effect and the spacing distance was also studied and some experimental conditions were optimized, on the basis of which a novel ET-based PEC TBP biosensor was realized with high sensitivity and selectivity.

Background Transcription factors (TFs) are effectors of cell signaling pathways that regulate gene expression. TF networks are highly interconnected; one signal can lead to changes in many TF levels, and one TF level can be changed by many different signals. TF regulation is central to normal cell function, with altered TF function being implicated in many disease conditions. Thus, measuring TF levels in parallel, and over time, is crucial for understanding the impact of stimuli on regulatory networks and on diseases. Results Here, we report the parallel analysis of temporal TF level changes due to multiple stimuli in distinct cell types. We have analyzed short-term dynamic changes in the levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), cAMP response element-binding protein (CREB), glucocorticoid receptor (GR), and TATA binding protein (TBP), in breast and liver cancer cells after tumor necrosis factor-alpha (TNF-α) and palmitic acid (PA) exposure. In response to both stimuli, NF-kB and CREB levels were increased, Stat3 decreased, and TBP was constant. GR levels were unchanged in response to TNF-α stimulation and increased in response to PA treatment. Conclusions Our results show significant overlap in signaling initiated by TNF-α and by PA, with the exception that the events leading to PA-mediated cytotoxicity likely also include induction of GR signaling. These results further illuminate the dynamics of TF responses to cytokine and fatty acid exposure, while concomitantly demonstrating the utility of parallel TF measurement approaches in the analysis of biological phenomena. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0293-6) contains supplementary material, which is available to authorized users.

TATA binding protein (TBP) is a key component of the eukaryotic RNA polymerase II transcription machinery that binds to TATA boxes located in the core promoter regions of many genes. Structural and biochemical studies have shown that when TBP binds DNA, it sharply bends the DNA. We used single-molecule fluorescence resonance energy transfer (smFRET) to study DNA bending by human TBP on consensus and mutant TATA boxes in the absence and presence of TFIIA. We found that the state of the bent DNA within populations of TBP-DNA complexes is homogeneous; partially bent intermediates were not observed. In contrast to the results of previous ensemble studies, TBP was found to bend a mutant TATA box to the same extent as the consensus TATA box. Moreover, in the presence of TFIIA, the extent of DNA bending was not significantly changed, although TFIIA did increase the fraction of DNA molecules bound by TBP. Analysis of the kinetics of DNA bending and unbending revealed that on the consensus TATA box two kinetically distinct populations of TBP-DNA complexes exist; however, the bent state of the DNA is the same in the two populations. Our smFRET studies reveal that human TBP bends DNA in a largely uniform manner under a variety of different conditions, which was unexpected given previous ensemble biochemical studies. Our new observations led to us to revise the model for the mechanism of DNA binding by TBP and for how DNA bending is affected by TATA sequence and TFIIA.

The autosomal dominant cerebellar ataxias (ADCAs) represent a heterogeneous group of neurodegenerative diseases with progressive ataxia and cerebellar degeneration. The current classification of this disease group is based on the underlying genetic defects and their typical disease courses. According to this categorization, ADCAs are divided into the spinocerebellar ataxias (SCAs) with a progressive disease course, and the episodic ataxias (EA) with episodic occurrences of ataxia. The prominent disease symptoms of the currently known and genetically defined 31 SCA types result from damage to the cerebellum and interconnected brain grays and are often accompanied by more specific extra-cerebellar symptoms. In the present review, we report the genetic and clinical background of the known SCAs and present the state of neuropathological investigations of brain tissue from SCA patients in the final disease stages. Recent findings show that the brain is commonly seriously affected in the polyglutamine SCAs (i.e. SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17) and that the patterns of brain damage in these diseases overlap considerably in patients suffering from advanced disease stages. In the more rarely occurring non-polyglutamine SCAs, post-mortem neuropathological data currently are scanty and investigations have been primarily performed in vivo by means of MRI brain imaging. Only a minority of SCAs exhibit symptoms and degenerative patterns allowing for a clear and unambiguous diagnosis of the disease, e.g. retinal degeneration in SCA7, tau aggregation in SCA11, dentate calcification in SCA20, protein depositions in the Purkinje cell layer in SCA31, azoospermia in SCA32, and neurocutaneous phenotype in SCA34. The disease proteins of polyglutamine ataxias and some non-polyglutamine ataxias aggregate as cytoplasmic or intranuclear inclusions and serve as morphological markers. Although inclusions may impair axonal transport, bind transcription factors, and block protein quality control, detailed molecular and pathogenetic consequences remain to be determined.

The autosomal dominant cerebellar ataxias (ADCAs) represent a heterogeneous group of neurodegenerative diseases with progressive ataxia and cerebellar degeneration. The current classification of this disease group is based on the underlying genetic defects and their typical disease courses. According to this categorization, ADCAs are divided into the spinocerebellar ataxias (SCAs) with a progressive disease course, and the episodic ataxias (EA) with episodic occurrences of ataxia. The prominent disease symptoms of the currently known and genetically defined 31 SCA types result from damage to the cerebellum and interconnected brain grays and are often accompanied by more specific extra-cerebellar symptoms. In the present review, we report the genetic and clinical background of the known SCAs and present the state of neuropathological investigations of brain tissue from SCA patients in the final disease stages. Recent findings show that the brain is commonly seriously affected in the polyglutamine SCAs (i.e. SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17) and that the patterns of brain damage in these diseases overlap considerably in patients suffering from advanced disease stages. In the more rarely occurring non-polyglutamine SCAs, post-mortem neuropathological data currently are scanty and investigations have been primarily performed in vivo by means of MRI brain imaging. Only a minority of SCAs exhibit symptoms and degenerative patterns allowing for a clear and unambiguous diagnosis of the disease, e.g. retinal degeneration in SCA7, tau aggregation in SCA11, dentate calcification in SCA20, protein depositions in the Purkinje cell layer in SCA31, azoospermia in SCA32, and neurocutaneous phenotype in SCA34. The disease proteins of polyglutamine ataxias and some non-polyglutamine ataxias aggregate as cytoplasmic or intranuclear inclusions and serve as morphological markers. Although inclusions may impair axonal transport, bind transcription factors, and block protein quality control, detailed molecular and pathogenetic consequences remain to be determined.

TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G1 phase cells. TLP stimulated expression of several growth-related genes including p21 (p21Waf1/Cip1). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.

Single-nucleotide polymorphisms (SNPs) in the regulatory regions of human genes and, in particular, TATA boxes can alter the sensitivity to bacterial and viral infections and to drugs used for their treatment. These SNPs are associated with the risk of widespread multifactorial human diseases, such as arthritis, hypertension, cancer, Alzheimer’s disease, and others. Interaction of the TATA-binding protein (TBP) with TATA boxes on TATA-containing promoters nucleates the preinitiation complex assemblage, which is the initial stage of transcription regulation. This study considers the change of TBP affinity to the TATA boxes of human MBL, CYP 2A6, SOD1, and TPI genes bearing SNPs associated with autoimmune diseases, cardiovascular diseases, amyotrophic lateral sclerosis, neurodegenerative disorders, hypersensitivity to bacterial infections, etc. In all cases a decrease in the affinity of TBP to TATA-containing oligonucleotides was shown.

To investigate the function and architecture of the open complex state of RNA polymerase II (Pol II), Saccharomyces cerevisiae minimal open complexes were assembled by using a series of heteroduplex HIS4 promoters, TATA binding protein (TBP), TFIIB, and Pol II. The yeast system demonstrates great flexibility in the position of active open complexes, spanning 30 to 80 bp downstream from TATA, consistent with the transcription start site scanning behavior of yeast Pol II. TFIIF unexpectedly modulates the activity of the open complexes, either repressing or stimulating initiation. The response to TFIIF was dependent on the sequence of the template strand within the single-stranded bubble. Mutations in the TFIIB reader and linker region, which were inactive on duplex DNA, were suppressed by the heteroduplex templates, showing that a major function of the TFIIB reader and linker is in the initiation or stabilization of single-stranded DNA. Probing of the architecture of the minimal open complexes with TFIIB-FeBABE [TFIIB-p-bromoacetamidobenzyl-EDTA-iron(III)] derivatives showed that the TFIIB core domain is surprisingly positioned away from Pol II, and the addition of TFIIF repositions the TFIIB core domain to the Pol II wall domain. Together, our results show an unexpected architecture of minimal open complexes and the regulation of activity by TFIIF and the TFIIB core domain.

Background Obesity affects quality of life and life expectancy and is associated with cardiovascular disorders, cancer, diabetes, reproductive disorders in women, prostate diseases in men, and congenital anomalies in children. The use of single nucleotide polymorphism (SNP) markers of diseases and drug responses (i.e., significant differences of personal genomes of patients from the reference human genome) can help physicians to improve treatment. Clinical research can validate SNP markers via genotyping of patients and demonstration that SNP alleles are significantly more frequent in patients than in healthy people. The search for biomedical SNP markers of interest can be accelerated by computer-based analysis of hundreds of millions of SNPs in the 1000 Genomes project because of selection of the most meaningful candidate SNP markers and elimination of neutral SNPs. Results We cross-validated the output of two computer-based methods: DNA sequence analysis using Web service SNP_TATA_Comparator and keyword search for articles on comorbidities of obesity. Near the sites binding to TATA-binding protein (TBP) in human gene promoters, we found 22 obesity-related candidate SNP markers, including rs10895068 (male breast cancer in obesity); rs35036378 (reduced risk of obesity after ovariectomy); rs201739205 (reduced risk of obesity-related cancers due to weight loss by diet/exercise in obese postmenopausal women); rs183433761 (obesity resistance during a high-fat diet); rs367732974 and rs549591993 (both: cardiovascular complications in obese patients with type 2 diabetes mellitus); rs200487063 and rs34104384 (both: obesity-caused hypertension); rs35518301, rs72661131, and rs562962093 (all: obesity); and rs397509430, rs33980857, rs34598529, rs33931746, rs33981098, rs34500389, rs63750953, rs281864525, rs35518301, and rs34166473 (all: chronic inflammation in comorbidities of obesity). Using an electrophoretic mobility shift assay under nonequilibrium conditions, we empirically validated the statistical significance (α < 0.00025) of the differences in TBP affinity values between the minor and ancestral alleles of 4 out of the 22 SNPs: rs200487063, rs201381696, rs34104384, and rs183433761. We also measured half-life (t1/2), Gibbs free energy change (ΔG), and the association and dissociation rate constants, ka and kd, of the TBP-DNA complex for these SNPs. Conclusions Validation of the 22 candidate SNP markers by proper clinical protocols appears to have a strong rationale and may advance postgenomic predictive preventive personalized medicine.

Studying the regulatory mechanism of the glycoprotein hormone α subunit (αGSU) gene in thyrotropes is essential for understanding the synthesis of functional thyroid-stimulating hormone (TSH). Here, we investigated the influence of a homeodomain transcription factor Msx1 (Msh homeobox 1) on αGSU expression in thyrotropes. The transient expression of Msx1 inhibited the activity of an αGSU reporter gene, as well as its endogenous mRNA level in thyrotrope-derived αTSH cells. Luciferase reporter assays with serial deletion constructs and a close examination of the sequences revealed that the putative Msx1 binding site (PMS) in the αGSU promoter is not responsible for Msx1-mediated transcriptional repression. We also identified the TATA-box binding protein (TBP) as an interacting protein in thyrotropes. Interaction of TBP with Msx1 attenuates the inhibitory effect of Msx1 on αGSU gene expression in a DNA binding-independent manner. Furthermore, transient transfection studies with mutant Msx1 revealed that the interaction of TBP and Msx1 is critical for Msx1-mediated transcriptional repression of the αGSU. These results suggest that Msx1 functions as a transcriptional repressor of αGSU and that its interaction with TBP is an integral part of the mechanism by which Msx1 regulates the inhibition of αGSU gene expression.

The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells.

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.

The development of convenient, real-time probes for monitoring protein function in biological samples represents an important challenge of the postgenomic era. In response, we introduce here “transcription factor beacons,” binding-activated fluorescent DNA probes that signal the presence of specific DNA-binding activities. As a proof of principle, we present beacons for the rapid, sensitive detection of three transcription factors (TATA Binding Protein, Myc-Max, and NF-κB), and measure binding activity directly in crude nuclear extracts.

The Swi2/Snf2 family of proteins uses the energy of ATP hydrolysis to disrupt protein-DNA interactions, a process known as remodelling. In this study, Karl-Peter Hopfner and colleagues have solved the structure of the Mot1 ATPase in complex with the transcription factor TBP, which Mot1 displaces from promoters. The structure reveals how the ATPase specifically interacts with TBP and DNA, acting both to displace TBP and to prevent it from rebinding DNA. Swi2/Snf2-type ATPases regulate genome-associated processes such as transcription, replication and repair by catalysing the disruption, assembly or remodelling of nucleosomes or other protein–DNA complexes1,2. It has been suggested that ATP-driven motor activity along DNA disrupts target protein–DNA interactions in the remodelling reaction3,4,5. However, the complex and highly specific remodelling reactions are poorly understood, mostly because of a lack of high-resolution structural information about how remodellers bind to their substrate proteins. Mot1 (modifier of transcription 1 in Saccharomyces cerevisiae, denoted BTAF1 in humans) is a Swi2/Snf2 enzyme that specifically displaces the TATA box binding protein (TBP) from the promoter DNA and regulates transcription globally by generating a highly dynamic TBP pool in the cell6,7. As a Swi2/Snf2 enzyme that functions as a single polypeptide and interacts with a relatively simple substrate, Mot1 offers an ideal system from which to gain a better understanding of this important enzyme family. To reveal how Mot1 specifically disrupts TBP–DNA complexes, we combined crystal and electron microscopy structures of Mot1–TBP from Encephalitozoon cuniculi with biochemical studies. Here we show that Mot1 wraps around TBP and seems to act like a bottle opener: a spring-like array of 16 HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats grips the DNA-distal side of TBP via loop insertions, and the Swi2/Snf2 domain binds to upstream DNA, positioned to weaken the TBP–DNA interaction by DNA translocation. A ‘latch’ subsequently blocks the DNA-binding groove of TBP, acting as a chaperone to prevent DNA re-association and ensure efficient promoter clearance. This work shows how a remodelling enzyme can combine both motor and chaperone activities to achieve functional specificity using a conserved Swi2/Snf2 translocase.

Neuropeptide Y (NPY) is a potent orexigenic agent. The molecular mechanisms underlying the regulation of bovine NPY gene expression by its promoter region is currently unknown. The objectives of this research were to: (i) identify the SNPs in the promoter region of the bovine NPY gene, (ii) investigate the effects of these SNPs by measuring promoter transcriptional activities of different bovine NPY promoter haplotypes and; (iii) identify the minimal promoter region (MPR) required for basal activity of the NPY gene in vitro. Seventeen SNPs were identified in the promoter region. Of these, 14 affected putative transcription factors binding motifs including a TATA binding protein factor at -20, GC-Box factors SP1 at -170 and GATA binding motifs at -120 and -347. The SNPs were assigned to five major haplotypes (BtNPY_H1-5), of which BtNPY_H5 had maximum transcriptional activity. The region extending to -134 nt was identified as the MPR. This MPR was confirmed by the identification of a putative TATA box (-29 nt) and two SP1/GC binding sites (-94 and -118 nt), within this region. However, promoter expression was significantly enhanced when the construct contained the -614 to -1019 nt region. In conclusion, a number of SNPs characterised in the bovine NPY promoter especially those affecting the transcription factor binding sites, enhancer and repressor regions have the potential to affect NPY gene expression. Natural variation exists in the promoter region of the bovine NPY gene, which should be further explored for selection of energetic efficiency in cattle.

The UBE2C oncogene is overexpressed in many types of solid tumours including the lethal castration-resistant prostate cancer (CRPC). The underlying mechanisms causing UBE2C gene overexpression in CRPC are not fully understood. Here, we show that CRPC-specific enhancers drive UBE2C overexpression in both AR-negative and -positive CRPC cells. We further show that co-activator MED1 recruitment to the UBE2C enhancers is required for long-range UBE2C enhancer/promoter interactions. Importantly, we find that the molecular mechanism underlying MED1-mediated chromatin looping involves PI3K/AKT phosphorylated MED1-mediated recruitment of FoxA1, RNA polymerase II and TATA binding protein and their subsequent interactions at the UBE2C locus. MED1 phosphorylation leads to UBE2C locus looping, UBE2C gene expression and cell growth. Our results not only define a causal role of a post-translational modification (phosphorylation) of a co-activator (MED1) in forming or sustaining an active chromatin structure, but also suggest that development of specific therapies for CRPC should take account of targeting phosphorylated MED1.

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variability in humans. Hereditary disorders in the hemoglobin synthesis caused by SNPs in the TATA boxes of the human β-globin gene promoter are associated with β-thalassemia of varying severity. The TATA-binding protein (TBP) is the first basal transcription factor that recognizes and binds TATA boxes in TATA-box-containing promoters and initiates the assembly of RNA polymerase II transcription complexes. This report is the first to present quantitative parameters (Kd) of human TBP interaction with SNP-containing TATA boxes of the β-globin gene promoter. These data are in agreement with literature data on the decrease in normal β-globin RNA synthesis in patients with β-thalassemia.

The general Transcription Factor IID is a key player in the early events of gene expression. TFIID is a multisubunit complex composed of the TATA Binding Protein and at least thirteen TBP Associated Factors (TAfs) which recognize the promoter of protein coding genes in a activator dependant way. This review highlights recent findings on the molecular architecture and dynamics of TFIID. The structural analysis of functional transcription complexes formed by TFIID, TFIIA, activators and/or promoter DNA illuminates the faculty of TFIID to adjust to various promoter architectures and highlights its role as a platform for preinitiation complex assembly.

// Jose M. Morachis 1,2 , Ruo Huang 1 , Beverly M. Emerson 1 1 Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA 2 Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA Keywords: RNA polymerase II, transcription therapy, core promoters, kinase inhibitors, p53, p21, Fas/APO1 Received: January 21, 2011; Accepted: January 25, 2010; Published: January 27, 2010; Correspondence: Beverly M. Emerson, e-mail: // Abstract Our current understanding of eukaryotic transcription has greatly benefited from use of small molecule inhibitors that have delineated multiple regulatory steps in site-specific initiation and elongation of RNA synthesis by multiple forms of RNA polymerase (RNAP). This class of "transcription" drugs is also of therapeutic interest and under evaluation in clinical trials. However, to date very few small molecules that directly abolish transcription have been identified, particularly those that act at the level of RNAP II initiation. Using a biochemical assay that measures transcription from recombinant, natural p53-responsive promoters and an artificial "super" promoter, we have identified three distinct small molecules that inhibit mRNA synthesis in vitro. Unexpectedly, these are kinase inhibitors, Hypericin, Rottlerin, and SP600125, with known substrates, which we find also strongly impair transcriptional initiation (IC50s = µM range) by targeting specific components of the RNAP II pre-initiation complex. When measured before and during transcription in vitro, one common target of inhibition by all three compounds is modification of the TATA Binding Protein (TBP) within the RNAP II holocomplex as it converts to an active transcribing enzyme. On this basis, by blocking the critical step of TBP modification, transcriptional initiation is effectively abolished even on structurally distinct core promoters.

The Arabidopsis thaliana trithorax-like protein, ATX1, shares common structural domains, has similar histone methyltransferase (HMT) activity, and belongs in the same phylogenetic subgroup as its animal counterparts. Most of our knowledge of the role of HMTs in trimethylating lysine 4 of histone H3 (H3K4me3) in transcriptional regulation comes from studies of yeast and mammalian homologs. Little is known about the mechanism by which ATX1, or any other HMT of plant origin, affects transcription. Here, we provide insights into how ATX1 influences transcription at regulated genes, playing two distinct roles. At promoters, ATX1 is required for TATA binding protein (TBP) and RNA Polymerase II (Pol II) recruitment. In a subsequent event, ATX1 is recruited by a phosphorylated form of Pol II to the +300-bp region of transcribed sequences, where it trimethylates nucleosomes. In support of this model, inhibition of phosphorylation of the C-terminal domain of Pol II reduced the amounts of H3K4me3 and ATX1 bound at the +300-nucleotide region. Importantly, these changes did not reduce the occupancy of ATX1, TBP, or Pol II at promoters. Our results indicate that ATX1 affects transcription at target genes by a mechanism distinct from its ability to trimethylate H3K4 within genes.

We in vitro examined the existing prognoses of the dissociation constant, KD, between ТАТА- Binding Protein (TBP) and ТАТА box with single nucleotide polymorphism (SNP) associated with human diseases. Five SNPs of the genes for cytochrome P450 2A6 (associated with lung cancer), β-globin (associated with β-thalassemia), mannose binding lectin (associated with variable immunodeficiency), superoxide dismutase 1 (associated with amyotrophic lateral sclerosis) and triosephosphate isomerase (associated with anemia) fell within the range of –ln(KD;M/KD;WT) between –1.5 and –1 (here KD;WT and KD;M denote the normal ТАТА box and with SNP). The mea-surements using EMSA demonstrated that: 1) all the predictions stating that the affinity between ТВР and ТАТА boxes with SNPs would be reduced were correct; 2) the departures of three predictions from the measurements fell within the confidence interval; 3) all the predictions consistently underestimated actual mutational damage caused to ТАТА boxes with SNPs (a < 0.05; binomial law) and two of these predictions did so significantly (a < 0.05, Student’s t-test). This consistent underestimation seems to be associated with the damage to the context that modulates ТВP/ТАТА affinity, for example, the contact between the nucleosomal histone H3-Н4 dimer and the core promoter immediately near ТАТА boxes.

The human cytomegalovirus (HCMV) IE86 protein is essential for HCMV replication due to its ability to transactivate critical viral early promoters. In the current study, we performed a comprehensive mutational analysis between amino acids (aa) 535 and 545 of IE86 and assessed the impact of these mutations on IE86-mediated transcriptional activation. Using transient assays and complementing analysis with recombinant HCMV clones, we show that single amino acid mutations differentially impair the ability of IE86 to mediate transactivation of essential early gene promoters. The conserved tyrosine at amino acid 544 is critical for activation of the UL54 promoter in vitro and in the context of the viral genome. In contrast, mutation of the proline at position 535 disrupted activation of the UL54 promoter in transient assays but displayed activity similar to that of wild-type (WT) IE86 when assessed in the genomic context. To examine the underlying mechanism of this differential effect, glutathione S -transferase (GST) pulldown assays were performed, revealing that Y544 is critical for binding to the TATA binding protein (TBP), suggesting that this interaction is likely necessary for the ability of IE86 to activate the UL54 promoter. In contrast, mutation of either P535 or Y544 disrupted activation of the UL112-113 promoter both in vitro and in vivo , suggesting that interaction with TBP is not sufficient for IE86-mediated activation of this early promoter. Together, these studies demonstrate that IE86 activates early promoters by distinct mechanisms.

The aryl hydrocarbon receptor (AhR) mediates induction of CYP1A1 and CYP1B1 by 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (dioxin) via binding to xenobiotic-responsive elements (XREs) in their enhancer regions. CYP1A1 and CYPIB1 were both inducible by dioxin in human MCF-7 cells. However, only CYP1A1 was inducible in human HepG2 cells. Further experiments focused on providing an explanation for this last observation. Dioxin induced the recruitment of AHR and the transcriptional coactivators p300 and p300/cAMP response element-binding protein binding protein-associated factor (PCAF) to the CYP1B1 enhancer in HepG2 cells but failed to induce recruitment of RNA polymerase II (polII) or the TATA binding protein (TBP) and acetylations of histones 3 and 4 or methylation of histone 3 at the promoter. Because p300 was required for dioxin induction of the aforementioned histone modifications at the CYP1B1 promoter and for induction of CYP1B1 transcription (in MCF-7 cells), the recruitments of p300 and AhR, although necessary, are not sufficient for eliciting the above responses to dioxin. Cytosine residues within CpG dinucleotides at the enhancer, including those within the XREs, were partially methylated, whereas those at the promoter were fully methylated. Treatment of HepG2 cells with 5-aza-2′-deoxycytidine led to partial demethylation of the promoter, restored polII and TBP binding, and CYP1B1 inducibility. Thus, the deficiency of CYP1B1 induction in HepG2 cells is ascribable to cytosine methylation at the promoter, which prevents recruitment of TBP and polII. It is noteworthy that our data indicate that stable recruitment of p300 and PCAF to the CYP1B1 gene does not require their tethering to the promoter and to the enhancer.

A novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNA-binding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. Interestingly, we disclosed a new finding that binding of target proteins to their binding sequences in the aggregated AuNP network rendered a stable and long-period protection of DNA. Unlike conventional fluorescence assays merely based on temporal protection of DNA from Exo III digestion, the stable protection afforded a static color transition indicator for DNA-protein interactions with no time-dependent monitoring required in the assay. Therefore, it furnished the developed strategy with improved technical robustness and operational convenience. Furthermore, we introduced thioctic acid as a stable anchor for tethering DNA on AuNPs. This DNA-tethering protocol circumvented the interferences from thiol compounds in common enzymatic systems. The Exo III protection-based colorimetric biosensor was demonstrated using a model target of TATA binding protein, a key transcriptional factor involving in various transcriptional regulatory networks. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM.

We studied expression of 90 miRNAs in ST Hdh Q111 / Hdh Q111 cells, a model for Huntington’s disease and compared with that obtained in ST Hdh Q7 / Hdh Q7 cells. Fifteen miRNAs were down regulated and 12 miRNAs were up regulated more than 2-fold. Such changes were statistically significant. One hundred and forty-two genes are experimentally known targets of these altered miRNAs. It has been predicted that miR-146a may target Tata Binding Protein ( TBP ). Using luciferase reporter assays with 3′-UTRs of TB P , over-expression and inhibition of miR-146a, we showed that miR-146a targets TBP . Regulation of TBP by miR-146a may contribute to HD pathogenesis.

Evolutionary trends have been examined in 146 HIV-1 forms (2662 copies, 2311 isolates) polymorphic for the TATA box using the "DNA sequence→affinity for TBP" regression (TBP is the TATA binding protein). As a result, a statistically significant excess of low-affinity TATA box HIV-1 variants corresponding to a low level of both basal and TAT-dependent expression and, consequently, slow replication of HIV-1 have been detected. A detailed analysis revealed that the excess of slowly replicating HIV-1 is associated with the subtype E-associated TATA box core sequence "CATAAAA". Principal Component Analysis performed on 2662 HIV-1 TATA box copies in 70 countries revealed the presence of two principal components, PC1 (75.7% of the variance) and PC2 (23.3% of the variance). They indicate that each of these countries is specifically associated with one of the following trends in HIV-1 evolution: neutral drift around the normal TATA box; neutral drift around the slowly replicating TATA box core sequence (phylogenetic inertia); an adaptive increase in the frequency of the slowly replicating form.

Motivation: ChIP-chip and ChIP-seq technologies provide genome-wide measurements of various types of chromatin marks at an unprecedented resolution. With ChIP samples collected from different tissue types and/or individuals, we can now begin to characterize stochastic or systematic changes in epigenetic patterns during development (intra-individual) or at the population level (inter-individual). This requires statistical methods that permit a simultaneous comparison of multiple ChIP samples on a global as well as locus-specific scale. Current analytical approaches are mainly geared toward single sample investigations, and therefore have limited applicability in this comparative setting. This shortcoming presents a bottleneck in biological interpretations of multiple sample data. Results: To address this limitation, we introduce a parametric classification approach for the simultaneous analysis of two (or more) ChIP samples. We consider several competing models that reflect alternative biological assumptions about the global distribution of the data. Inferences about locus-specific and genome-wide chromatin differences are reached through the estimation of multivariate mixtures. Parameter estimates are obtained using an incremental version of the Expectation–Maximization algorithm (IEM). We demonstrate efficient scalability and application to three very diverse ChIP-chip and ChIP-seq experiments. The proposed approach is evaluated against several published ChIP-chip and ChIP-seq software packages. We recommend its use as a first-pass algorithm to identify candidate regions in the epigenome, possibly followed by some type of second-pass algorithm to fine-tune detected peaks in accordance with biological or technological criteria. Availability: R source code is available at http://gbic.biol.rug.nl/supplementary/2009/ChromatinProfiles/ Access to Chip-seq data: GEO repository GSE17937 Contact: f.johannes@rug.nl Supplementary information: Supplementary data are available at Bioinformatics online.

The basal RNA polymerase II (RNAPII) transcription machinery is composed of RNAPII and the general transcription factors (TF) TATA binding protein (TBP), TFIIB, TFIIE, TFIIF and TFIIH. Due to the powerful genetic and molecular approaches that can be utilized, the budding yeast Saccharomyces cerevisiae has proven to be an invaluable model system for studies of the mechanisms of RNAPII transcription. Complementary biochemical studies of the S. cerevisiae basal transcription machinery, however, have been hampered by difficulties in the purification of TFIIF and TFIIH, most notably due to the severe toxicity of the TFIIF Tfg1 subunit in Escherichia coli and the complexity of the purification scheme for native TFIIH. Here, we report the elimination of TFG1-associated toxicity in E. coli, the identification and removal of a functional E. coli promoter and internal translation initiation within the N-terminal coding region of TFG1, and the efficient production and two-step purification of recombinant TFIIF complexes. We also report conditions for the efficient two-step tandem affinity purification (TAP) of holo-TFIIH, core TFIIH and TFIIK complexes from yeast whole cell extracts.

Recent findings associate transcription start in trypanosomatids with chromatin regions containing modified and variant histones. TATA-binding protein (TBP) and other fundamental transcription factors have been also found at these Transcription Start Sites (TSS). Results of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments show that Trypanosoma cruzi TBP (TcTBP) has an in vitro binding preference for G-rich sequences. This finding correlates with the presence of G-rich stretches at the Strand Switch Regions (SSR) and at some putative internal TSS in Trypanosoma brucei and Leishmania. A scanning study of partially assembled T. cruzi genomic contigs determined the presence of G-rich stretches in the coding strands. TcTBP affinity for G-rich sequences suggests that this factor could play a role in locating the initiation complex in the right TSS, probably by "sensing" the G-content on the strand to be transcribed.

African swine fever virus (ASFV) is the etiological agent of ASF, a fatal hemorrhagic fever that affects domestic pigs. There is currently no vaccine against ASFV, making it a significant threat to the pork industry. The ASFV genome sequence has been published; however, about half of ASFV open reading frames have not been characterized in terms of their structure and function despite being essential for our understanding of ASFV pathogenicity. The present study reports the three-dimensional structure and function of uncharacterized protein, pB263R (NP_042780.1), an open reading frame found in all ASFV strains. Sequence-based profiling and hidden Markov model search methods were used to identify remote pB263R homologs. Iterative Threading ASSEmbly Refinement (I-TASSER) was used to model the three-dimensional structure of pB263R. The posterior probability of fold family assignment was calculated using TM-fold, and biological function was assigned using TM-site, RaptorXBinding, Gene Ontology, and TM-align. Our results suggests that pB263R has the features of a TATA-binding protein and is thus likely to be involved in viral gene transcription.

Accumulating evidence indicates that TBP (TATA-binding protein)-like protein (TLP) contributes to the regulation of stress-mediated cell cycle checkpoint and apoptotic pathways, although its physiological target genes have remained elusive. In the present study, we have demonstrated that human TAp63 is one of the direct transcriptional target genes of TLP. Enforced expression of TLP results in the transcriptional induction of the endogenous TAp63, but not of the other p53 family members such as TAp73 and p53. Consistent with these results, small interference RNA-mediated knockdown led to a significant down-regulation of the endogenous TAp63. Luciferase reporter assay and chromatin immunoprecipitation analysis revealed that the genomic region located at positions -487 to -29, where +1 represents the transcriptional initiation site of TAp63, is required for TLP-dependent transcriptional activation of TAp63 and also TLP is efficiently recruited onto this region. Additionally, cells treated with anti-cancer drug etoposide underwent apoptosis in association with the transcriptional enhancement of TAp63 in a p53-independent manner, and the knockdown of the endogenous TLP reduced etoposide-induced apoptosis through repression of TAp63 expression. Taken together, our present study identifies a TLP-TAp63 pathway that is further implicated in stress-induced apoptosis.

Electrochemical impedance probing of TATA binding protein (TBP) based on TATA box site-specific binding was described in this work. A sensitive detection of TBP was developed from TATA box DNA self assembly on the electrode and the impedance changes induced by TBP binding. Electrochemical impedance spectroscopy (EIS) probing of TBP had a sensitivity of 0.8 nM with excellent selectivity. Moreover, the interferences of triplex forming oligonucleotides (TFOs) and anticancer drug daunomycin on TBP binding to TATA box DNA were investigated by EIS. TFOs reduced the stability of TBP binding to TATA box, but daunomycin completely inhibited the TBP binding. Keywords: TATA binding protein, Electrochemical impedance spectroscopy, DNA, Triplex formation oligonucleotides

Eukaryotic GCN5 acetyltransferases influence diverse biological processes by acetylating histones and non-histone proteins and regulating chromatin and gene-specific transcription as part of multiprotein complexes. In lower eukaryotes and invertebrates, these complexes include the yeast ADA complex that is still incompletely understood; the SAGA (Spt-Ada-Gcn5 acetylase) complexes from yeast to Drosophila that are mostly coactivators; and the ATAC (Ada Two-A containing) complex, only known in Drosophila and still poorly characterized. In contrast, vertebrate organisms, express two paralogous GCN5-like acetyltransferases (GCN5 and PCAF), which have been found so far only in SAGA-type complexes referred to hereafter as the STAGA (SPT3-TAF9-GCN5/PCAF acetylase) complexes. We now report the purification and characterization of vertebrate (human) ATAC-type complexes and identify novel components of STAGA. We show that human ATAC complexes incorporate in addition to GCN5 or PCAF (GCN5/PCAF), other epigenetic coregulators (ADA2-A, ADA3, STAF36, and WDR5), cofactors of chromatin assembly/remodeling and DNA replication machineries (POLE3/CHRAC17 and POLE4), the stress- and TGFβ-activated protein kinase (TAK1/MAP3K7) and MAP3-kinase regulator (MBIP), additional cofactors of unknown function, and a novel YEATS2-NC2β histone fold module that interacts with the TATA-binding protein (TBP) and negatively regulates transcription when recruited to a promoter. We further identify the p38 kinase-interacting protein (p38IP/FAM48A) as a novel component of STAGA with distant similarity to yeast Spt20. These results suggest that vertebrate ATAC-type and STAGA-type complexes link specific extracellular signals to modification of chromatin structure and regulation of the basal transcription machinery.

The TATA-binding protein (TBP) is critical for transcription by all three nuclear RNA polymerases. In order to identify factors that interact with TBP, the nonnatural photoreactive amino acid ρ-benzoyl-phenylalanine (BPA) was substituted onto the surface of Saccharomyces cerevisiae TBP in vivo. Cross-linking of these TBP derivatives in isolated transcription preinitiation complexes or in living cells reveals physical interactions of TBP with transcriptional coregulator subunits and with the general transcription factor TFIIA. Importantly, the results show a direct interaction between TBP and the SAGA coactivator subunits Spt3 and Spt8. Mutations on the Spt3-interacting surface of TBP significantly reduce the interaction of TBP with SAGA, show a corresponding decrease in transcription activation, and fail to recruit TBP to a SAGA-dependent promoter, demonstrating that the direct interaction of these factors is important for activated transcription. These results prove a key prediction of the model for stimulation of transcription at SAGA-dependent genes via Spt3. Our cross-linking data also significantly extend the known surfaces of TBP that directly interact with the transcriptional regulator Mot1 and the general transcription factor TFIIA.

We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein-protein photocrosslinking, and we have applied the approach to the analysis of interactions of alpha-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA-TBP-DNA complex and in higher order transcription-initiation complexes, and we have mapped the crosslink to the 'connector' region of the TFIIA alpha/beta subunit (TFIIAalpha/beta). We further have found that TBP H2 can be crosslinked to NC2 in the NC2-TBP-DNA complex, and we have mapped the crosslink to the C-terminal 'tail' of the NC2 alpha-subunit (NC2alpha). Interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail were not observed in crystal structures of TFIIA-TBP-DNA and NC2-TBP-DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP-TFIIA interactions and TBP-NC2 interactions, and provide an explanation-steric exclusion-for competition between TFIIA and NC2.

An undergraduate biochemistry laboratory experiment that will teach the technique of fluorescence resonance energy transfer (FRET) while analyzing protein-induced DNA bending is described. The experiment uses the protein TATA binding protein (TBP), which is a general transcription factor that recognizes and binds specific DNA sequences known as TATA boxes. When TBP binds to a TATA box, it bends the DNA. Such bending will be detected using FRET to measure the distance between two fluorophores located on the ends of the DNA. When TBP binds and bends the DNA, the fluorophores move closer together, reflected by an increase in FRET. At the completion of the experiment, three parameters will be determined: 1) the efficiency of the FRET, 2) the end-to-end distance between the fluorophores, and 3) the angle at which TBP bends the DNA. In performing this experiment, students will be introduced to FRET, gain experience in quantitative biophysical measurements, and appreciate how a protein can induce a dramatic change in DNA conformation.

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Mot1p and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Mot1p, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)–chip for cells in exponential growth and during reprogramming of transcription. We find that TBP, NC2, and Mot1p colocalize at transcriptionally active pol II core promoters. Relative binding of NC2α and Mot1p is higher at TATA promoters, whereas NC2β has a preference for TATA-less promoters. In line with the ChIP–chip data, we isolated a stable TBP–NC2–Mot1p–DNA complex from chromatin extracts. ATP hydrolysis releases NC2 and DNA from the Mot1p–TBP complex. In vivo experiments indicate that promoter dissociation of TBP and NC2 is highly dynamic, which is dependent on Mot1p function. Based on these results, we propose that NC2 and Mot1p cooperate to dynamically restrict TBP activity on transcribed promoters.

We describe a rapid and effective procedure for purifying recombinant eukaryotic TATA binding protein (TBP) from Escherichia coli. The method employs an affinity ligand comprising glutathione-S-transferase fused to the carboxyl-terminal activation domain of the transcriptional activator VP16 and an amino-terminal domain (TAND2) of the yeast TBP-associated factor TAF1. TBP can be purified without the need for extrinsic affinity tags, subsequent proteolysis, or downstream clean-up steps. This TBP purification process is rapid (requiring about 4 h after bacterial harvest) and does not require sophisticated chromatographic equipment. The resulting material is monodisperse, structured, and functionally active. We demonstrate the efficacy of this method for purifying recombinant full-length or TBP core fragments encoded by yeast, humans and Arabidopsis.

Essentially all nuclear eukaryotic gene transcription depends upon the function of the transcription factor TATA-binding protein (TBP). Here we show that the abundant, multifunctional DNA binding transcription factor repressor activator protein Rap1p interacts directly with TBP. TBP-Rap1p binding occurs efficiently in vivo at physiological expression levels, and in vitro analyses confirm that this is a direct interaction. The DNA binding domains of the two proteins mediate interaction between TBP and Rap1p. TBP-Rap1p complex formation inhibits TBP binding to TATA promoter DNA. Alterations in either Rap1p or TBP levels modulate mRNA gene transcription in vivo. We propose that Rap1p represents a heretofore unrecognized regulator of TBP.

TATA-binding protein (TBP) is essential for eukaryotic gene transcription. Human TBP contains a polymorphic polyglutamine (polyQ) domain in its N terminus and a DNA-binding domain in its highly conserved C terminus. Expansion of the polyQ domain to >42 glutamines typically results in spinocerebellar ataxia type 17 (SCA17), a neurodegenerative disorder that resembles Huntington disease. Our recent studies have demonstrated that polyQ expansion causes abnormal interaction of TBP with the general transcription factor TFIIB and induces neurodegeneration in transgenic SCA17 mice (Friedman, M. J., Shah, A. G., Fang, Z. H., Ward, E. G., Warren, S. T., Li, S., and Li, X. J. (2007) Nat. Neurosci. 10, 1519–1528). However, it remains unknown how polyQ expansion influences DNA binding by TBP. Here we report that polyQ expansion reduces in vitro binding of TBP to DNA and that mutant TBP fragments lacking an intact C-terminal DNA-binding domain are present in transgenic SCA17 mouse brains. polyQ-expanded TBP with a deletion spanning part of the DNA-binding domain does not bind DNA in vitro but forms nuclear aggregates and inhibits TATA-dependent transcription activity in cultured cells. When this TBP double mutant is expressed in transgenic mice, it forms nuclear inclusions in neurons and causes early death. These findings suggest that the polyQ tract affects the binding of TBP to promoter DNA and that polyQ-expanded TBP can induce neuronal toxicity independent of its interaction with DNA.

The binding of herpes simplex virus type 1 ICP4, TATA-binding protein (TBP), and RNA polymerase II (polII) to the promoter regions of representative immediate-early (IE) (ICP0), early (E) (thymidine kinase [tk]), and late (L) (glycoprotein C [gC]) genes on the viral genome was examined as a function of time postinfection, viral DNA replication, cis -acting sites for TFIID in the tk and gC promoters, and genetic background of ICP4. The binding of TBP and polII to the IE ICP0 promoter was independent of the presence of ICP4, whereas the binding of TBP and polII to the tk and gC promoters occurred only when ICP4 also bound to the promoters, suggesting that the presence of ICP4 at the promoters of E and L genes in virus-infected cells is crucial for the formation of transcription complexes on these promoters. When the TATA box of the tk promoter or the initiator element (INR) of the gC promoter was mutated, a reduction in the amount of TBP and polII binding was observed. However, a reduction in the amount of ICP4 binding to the promoters was also observed, suggesting that the binding of TBP-containing complexes and ICP4 is cooperative. The binding of ICP4, TBP, and polII was also observed on the gC promoter at early times postinfection or when DNA synthesis was inhibited, suggesting that transcription complexes may be formed early on L promoters and that additional events or proteins are required for expression. The ability to form these early complexes on the gC promoter required the DNA-binding domain but in addition required the carboxyl-terminal 524 amino acids of ICP4, which is missing the virus n208. This region was not required to form TBP- and polII-containing complexes on the tk promoter. n208 activates E but not L genes during viral infection. These data suggest that a region of ICP4 may differentiate between forming TBP- and polII-containing complexes on E and L promoters.