TATA Motif Research Articles

In Silico Verification of Predicted Potential Promoter Sequences in the Rice (Oryza sativa) Genome.

The exact identification of promoter sequences remains a serious problem in computational biology, as the promoter prediction algorithms under development continue to produce false-positive results. Therefore, to fully assess the validity of predicted sequences, it is necessary to perform a comprehensive test of their properties, such as the presence of downstream transcribed DNA regions behind them, or chromatin accessibility for transcription factor binding. In this paper, we examined the promoter sequences of chromosome 1 of the rice Oryza sativa genome from the Database of Potential Promoter Sequences predicted using a mathematical algorithm based on the derivation and calculation of statistically significant promoter classes. In this paper TATA motifs and cis-regulatory elements were identified in the predicted promoter sequences. We also verified the presence of potential transcription start sites near the predicted promoters by analyzing CAGE-seq data. We searched for unannotated transcripts behind the predicted sequences by de novo assembling transcripts from RNA-seq data. We also examined chromatin accessibility in the region of the predicted promoters by analyzing ATAC-seq data. As a result of this work, we identified the predicted sequences that are most likely to be promoters for further experimental validation in an in vivo or in vitro system.

New Genomic Signals Underlying the Emergence of Human Proto-Genes.

De novo genes are novel genes which emerge from non-coding DNA. Until now, little is known about de novo genes’ properties, correlated to their age and mechanisms of emergence. In this study, we investigate four related properties: introns, upstream regulatory motifs, 5′ Untranslated regions (UTRs) and protein domains, in 23,135 human proto-genes. We found that proto-genes contain introns, whose number and position correlates with the genomic position of proto-gene emergence. The origin of these introns is debated, as our results suggest that 41% of proto-genes might have captured existing introns, and 13.7% of them do not splice the ORF. We show that proto-genes which emerged via overprinting tend to be more enriched in core promotor motifs, while intergenic and intronic genes are more enriched in enhancers, even if the TATA motif is most commonly found upstream in these genes. Intergenic and intronic 5′ UTRs of proto-genes have a lower potential to stabilise mRNA structures than exonic proto-genes and established human genes. Finally, we confirm that proteins expressed by proto-genes gain new putative domains with age. Overall, we find that regulatory motifs inducing transcription and translation of previously non-coding sequences may facilitate proto-gene emergence. Our study demonstrates that introns, 5′ UTRs, and domains have specific properties in proto-genes. We also emphasize that the genomic positions of de novo genes strongly impacts these properties.

Locating TATA Binding Protein in the RNA Polymerase I Pre‐Initiation Complex

Transcription of eukaryotic DNA into RNA is carried by three main RNA polymerases (Pols I–III). Pol I synthesizes ribosomal DNA (rDNA) into ribosomal RNA (rRNA), which has been directly linked to the rate of protein synthesis, cell growth and proliferation. The efficiency of Pols’ function is controlled by general transcription factors, which enable the enzyme to interact with promoter DNA at the initiation stage. In Saccharomyces cerevisiae, Pol I transcription initiation relies on four main factors, one of which is TATA Binding Protein (TBP). TBP is an essential universal transcription factor used by all three eukaryotic Pols. As its name eludes, TBP typically binds to TATA motifs found in promoter DNA. Interestingly, unlike Pol II and III, Pol I promoters are TATA‐less. Thus, unlike the location of TBPs in other PICs, the location and function of TBP within the Pol I PIC remains unclear.Current Cryo‐EM structures of CF‐DNA‐Pol I initiation complex leaves little room for TBP to bind to its “typical” Pols II/III position. Therefore, we hypothesize that TBP must occupy a unique location, putatively more upstream, within the Pol I PIC. Combining available structural data with previous crosslinking data of CF and TBP, we modeled the interaction between the two proteins with varying distance constraints. In addition, we analyzed TBPs location on the rDNA promoter by site‐directed hydroxyl radical cleavage experiment, known as FeBABE, where DNA phosphothioate modification probes were constructed based on our predicted models. Our results from these combinational methods elucidated that TBP locates more upstream within Pol I PIC.Support or Funding InformationThis work was a part of SUNY Upstate Medical School’s Summer Undergraduate Research Fellowship 2019 and was supported by National Cancer Institute (NCI), National Institute for Dental and Craniofacial Research (NIDCR), and Carol Baldwin Research Foundation.FeBABE site‐directed hydroxyl radical cleavage.A and B. Sites of FeBABE incorporation into rDNA promoter in clusters of 5 include the “typical” location of TBP (1), upstream of the CE (2), 5′ of CE (3), and within the CE (4). C. Western blot showed FeBABE specific cleavage products. Cleavages were observed when FeBABE was incorporated at all four positions. Most intense cleavages were observed when FeBABE is incorporated upstream of the CE. D. Quantitation of TBP cleavages.Figure 1

Expression Analysis of Genes Encoding NHX2 Antiporter and Subunit A of Vacuolar H+-ATPase Pump in Salt-Resistant and Salt-Sensitive Barley (Hordeum vulgare L.) Cultivars under Salt Stress

Salinity is one of the most important factors causing limiting growth aspects. Salinity tolerance is a multigenic trait activating mechanisms such as high H+ pumping activity, like NHX2 that is a tonoplast localized Na+/H+ exchanger and actively transfers the excess sodium ions from the cytoplasm into the vacuole. Vacuolar (v-) H+-ATPase pump is also a tonoplast localized, providing the ATP energy required for tonoplast antiporters. Evaluation of the expression level of HvNHX2 and subunit A of v-H+-ATPase (VHA-A) genes using real-time RT-PCR, in root and shoot tissue of salt-sensitive Reihan and salt-tolerant Afzal barley (Hordeum vulgare L.) cultivars under different salinity concentrations indicated that increasing the concentrations or duration of NaCl stress would decrease the K+/Na ratio that is a signal for cascade responses to the stress but fluctuated expression level of studied genes in some time point. However, promoter analysis of studied genes showed that both have CAAT, TATA and W-box promoter motifs which involve pathogen, salt and ABA-responsive signaling pathways but differed in TC-rich repeats, A-box, TCA-element and GARE-motif that may be one of the reason of observed different.

Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes.

The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.

Gene structure and cDNA sequence of 2-Cys peroxiredoxin in the harmful algal bloom species Chattonella marina and its gene transcription under different light intensities

ABSTRACTWe investigated the gene structure and predicted amino acid sequence of the antioxidant enzyme 2-Cys peroxiredoxin (2-Cys Prx) in the raphidophyte Chattonella marina, which is a harmful algal bloom (HAB) species. The open reading frame of 2-Cys Prx was 585 bp long and encoded a protein consisting of 195 amino acids. The putative amino acid sequence contained two cysteine residues located at the 49th and 170th amino acid positions from the N-terminal methionine residue. The sequence also possessed 2-Cys Prx characteristic motifs, F (FFYPLDFTFVCPTEI) and EVCP. The position of the 2-Cys Prx gene relative to several others (ycf59 – 2-CysPrx – rpl35 – rpl20) was the same as that found in the chloroplast genome in the raphidophyte Heterosigma akashiwo. Upstream of the 2-Cys Prx gene, possible TATA and GGA motifs recognized by nuclear-encoded plastid RNA polymerase (NEP), and a possible -10 box and -35 box recognized by plastid-encoded plastid RNA polymerase (PEP) were observed. We measured the transcript levels of 2-Cys Prx in C. marina cells grown under three different light intensities (0, 100, 1000 µmol photons m–2 s–1, 14-h light/8-h dark photoperiod) by quantitative PCR. The 2-Cys Prx transcript level in cells grown under the highest light intensity on day 3 was threefold that on day 0 but two lower light intensities resulted in relatively stable transcription levels. The 2-Cys Prx transcript level was significantly positively related to the H2O2 concentration per cell and the H2O2 scavenging activity per cell. These results suggest that C. marina 2-Cys Prx functions in the chloroplast and its transcription could be regulated by both NEP and PEP. Moreover, the 2-Cys Prx transcript level might increase to remove excessive H2O2 produced under strong light conditions in order to maintain cell proliferation activity.

TSS seq based core promoter architecture in blood feeding Tsetse fly (Glossina morsitans morsitans) vector of Trypanosomiasis.

BackgroundTranscription initiation regulation is mediated by sequence-specific interactions between DNA-binding proteins (transcription factors) and cis-elements, where BRE, TATA, INR, DPE and MTE motifs constitute canonical core motifs for basal transcription initiation of genes. Accurate identification of transcription start site (TSS) and their corresponding promoter regions is critical for delineation of these motifs. To this end, the genome scale analysis of core promoter architecture in insects has been confined to Drosophila. The recently sequenced Tsetse fly genome provides a unique opportunity to analyze transcription initiation regulation machinery in blood-feeding insects.ResultsA computational method for identification of TSS in newly sequenced Tsetse fly genome was evaluated, using TSS seq tags sampled from two developmental stages namely; larvae and pupae. There were 3134 tag clusters among which 45.4 % (1424) of the tag clusters mapped to first coding exons or their proximal predicted 5′UTR regions and 1.0 % (31) tag clusters mapping to transposons, within a threshold of 100 tags per cluster. These 1393 non transposon-derived core promoters had propensity for AT nucleotides. The −1/+1 and 1/+1 positions in D. melanogaster, and G. m. morsitans had propensity for CA and AA dinucleotides respectively. The 1393 tag clusters comprised narrow promoters (5 %), broad with peak promoters (23 %) and broad without peak promoters (72 %). Two-way motif co-occurrence analysis showed that the MTE-DPE pair is over-represented in broad core promoters. The frequently occurring triplet motifs in all promoter classes are the INR-MTE-DPE, TATA-MTE-DPE and TATA-INR-DPE. Promoters without the TATA motif had higher frequency of the MTE and INR motifs than those observed in Drosophila, where the DPE motif occur more frequently in promoters without TATA motif. Gene ontology terms associated with developmental processes were overrepresented in the narrow and broad with peak promoters.ConclusionsThe study has identified different motif combinations associated with broad promoters in a blood-feeding insect. In the case of TATA-less core promoters, G.m. morsitans uses the MTE to compensate for the lack of a TATA motif. The increasing availability of TSS seq data allows for revision of existing gene annotation datasets with the potential of identifying new transcriptional units.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1921-6) contains supplementary material, which is available to authorized users.

Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: A role for NF-Y in regulating basal transcription

Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5′-regulatory region of the SLC25A19 gene (1080bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5′- and 3′-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between −131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.

De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements

Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells.

Transcriptional regulation of the gilthead seabream (Sparus aurata) interleukin-6 gene promoter

Interleukin-6 (IL-6) has been identified and characterized from several fish species and its mRNA expression is induced by pathogen-associated molecular patterns (PAMPs) and cytokines in immune cells and tissues. However, the transcriptional regulation of the IL-6 gene in fish is not well understood. In the present study, we have cloned and sequenced a 1028 bp 5′-flanking DNA region from the IL-6 gene in seabream (Sparus aurata). Sequence analysis of the seabream IL-6 promoter (sbIL-6P) evidenced the presence of a conserved TATA motif and conserved response elements for NF-κB, C/EBPβ (NF-IL6), AP-1 and GRE, similar to other vertebrate IL-6 promoters. Functional characterization of sbIL-6P was performed by cloning sbIL-6P into a luciferase expression vector and by transfecting it into L6 muscle cells, a mammalian cell line shown previously to express IL-6 in response to pro-inflammatory stimuli. We show here that the activity of sbIL-6P was significantly induced by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), IL-6 and IL-2, as well as by lipopolysaccharide (LPS), but significantly repressed by dexamethasone. In addition, the stimulatory effects of TNFα on sbIL-6P activity appeared to be mediated by the NF-κB, p38 MAPK and JNK signaling pathways. Deletion analyses of sbIL-6P suggested that activation of sbIL-6P by TNFα and IL-6 required the presence of binding motifs present in the proximal promoter (−171 to −84) whereas activation by IL-2 required binding motifs present in the distal promoter (−1024 to −864). The results from this study indicate, for the first time in fish, that pro-inflammatory cytokines, LPS and glucocorticoids can regulate the activity of the IL-6 gene at a transcriptional level and identify important regions in its response to cytokines.

CMV promoter is repressed by p53 and activated by JNK pathway

Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.

欧美杨水分利用效率相关基因PdEPF1的克隆及表达

提高植物水分利用效率(WUE)是未来解决我国甚至世界干旱缺水的最重要手段之一。在对植物WUE的众多研究方法中,大多集中在生理手段,但通过分子生物学手段研究其表达调控机制的较少。欧美杨(<em>Populus deltoides</em>×<em>Populus nigra</em>)是中纬度地区最适合种植的短轮伐期工业用材集约经营树种之一。近年来我国引进了许多优良的欧美杨无性系用于营造大面积的速生丰产林并取得很好的经济和社会效益。但高耗水量的缺点限制了其进一步的推广。通过基因芯片技术从欧美杨中找到一个可能调控WUE的基因-<em>PdEPF1</em>. 荧光定量表达进一步验证了这一结果。荧光定量表达分析表明该基因受ABA、盐、冷、干旱等胁迫诱导表达。组织特异表达分析说明<em>PdEPF1</em>基因在顶端叶和根中有表达,成熟叶衰老叶中则无表达。克隆到启动子分析表明该启动子含有多种干旱响应元件(drought response elememt),赤霉素响应元件(GA response elememt),低温响应元件(cold response elememt),ABA响应元件(ABA response elememt)等逆境相关的作用元件。;Water-use efficiency (WUE) is crucial to plant survival, yield and vegetation dynamics. Many researches trended to reduce water use by more crop per drop. Althoughmuch progresses have been achieved in determining the physiological mechanisms of WUE, there is few report on the genetic controls of WUE. The first gene ERECTA, which t promotes WUE, was shown to decrease stomatal density and be involved in the positive regulation of photosynthetic capacity.<br> The EPIDERMAL PATTERNING FACTOR 1 (EPF1) gene identified in Arabidopsis is expressed specifically in stomatal cells and precursors. EPF1 controls stomatal patterning through the regulation of asymmetric cell division, its activity depends on the TOO MANY MOUTHS receptor-like protein and ERECTA family receptor kinases, suggesting that EPF1 provides a positional cue interpreted by these receptors. A mutation of EPF1 results in increased stomatal density and violation of the one-cell-spacing rule (clustering of stomata). However, there is no report on the relationship between AtEPF1 and WUE.<br> The cottonwood poplar, Populus deltoides×Populus nigra, is one of the most important species in large-scale forestation in China. However, its high water consumption is a key factor limiting its growth in arid and semiarid regions. Identifcation of genes increasing WUE in this species is important for understanding stress responses and improvement of this species.<br> In this study, microarray analysis between poplar genotypes NE-19 (<em>P. nigra</em>×(<em>P. deltoides</em>×<em>P. nigra</em>)) with the higher WUE and DN-2 (<em>P. deltoides×P. nigra</em>) with a lower WUE,was performed. PdEPF1 from NE-19,one of the WUE-related genes, was identified, strongly expressed in immature leaves and roots, but not in senescent leaves, functional leaves and the stem. This gene is strongly induced by drought, abscisic acid and high salt levels, and enhanced by gibberellic acid. To unvarel the mechanism responsible for the inducible expression of PdEPF1 by different stresses, a 1090-bp PdEPF1 promoter fragment was isolated by the adaptor PCR with reference to the transcriptional start site determined from the PdEPF1 cDNA sequence. The PdEPF1 promoter contains CAAT and TATA motifs located at nucleotides -204 and -35 relative to the transcriptional start site, respectively. Each of these motifs has characteristics of eukaryotic gene promoters. Interestingly,, the 1090-bp promoter region contained several motifs based on the computer prediction, such as a W-box (a GA-responsive element), an MYC binding site, an early response to dehydration binding site, and a controlling guard cell-specific gene expression binding site. This indicate, that the stress-inducible expression of PdEPF1 is regulated at the level of transcription.

Optimization of reporter gene architecture for quantitative measurements of gene expression in the Drosophila embryo

Quantitative assessment of gene regulation is critical for mathematical modeling of transcriptional systems for systems biology efforts. Enhancers, also termed cis-regulatory modules (CRMs), are the primary mediators of transcriptional regulation in higher eukaryotes; transcription factors binding to CRMs dictate the likelihood and frequency of promoter activation. To provide a suitable platform for in-depth CRM analysis, we adapted a targeted integration vector to compare action of basal promoters with diverse combination of TATA, Inr and DPE motifs, as well as a set of 3'-UTRs representative of those used in different reporter vectors. This "Honda" series of reporter gene vectors was activated by a regulatory element binding Dorsal and Twist activators suitable for transcription in the early Drosophila embryo. The diverse promoters functioned in a similar manner with minor quantitative differences, consistent with a lack of enhancer-promoter specificity. Constructs bearing SV40 3'-UTR sequences appeared to produce somewhat higher levels of mRNA. Confocal laser scanning microscopy revealed that the mRNA distribution produced by these constructs was punctate; this pattern appears to be dependent on 5'-UTR sequences, as an optimized vector including an alternate 5'-UTR produced a more even distribution, which may be preferable for quantitative imaging. This set of Honda vectors contains convenient sites for modification of basal promoter, 3' UTR, and enhancer, and will be useful for analysis of CRMs and quantitative studies of gene expression.

Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation

In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5′-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5′-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB–DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5′-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues.

Genome-wide detection of a TFIID localization element from an initial human disease mutation

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17 181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.

Evolution of Repeated Sequences in the Ribosomal DNA Intergenic Spacer of 32 Arthropod Species

The evolution of a multigene family (MGF) is affected by the structure and function of its regulatory elements, specifically by the link between recombination and DNA transcription and/or replication. The ribosomal DNA (rDNA) MGF is often hierarchically repetitive, combining function with repetition in a single genic system. Its tandemly repeated operons contain the transcription unit of the 45S ribosomal RNA precursor alternating with an intergenic spacer (IGS) that commonly includes repeated transcription regulatory elements. To study the evolution of repeated sequences and the influence of repeat characteristics on their sequence divergence, we sequenced and characterized a single complete IGS from 11 daphniid species and analyzed their repeat arrays along with those from an additional 21 species of arthropods. We tested the hypotheses that sequence similarity is higher among tandemly arrayed repeats than among interleaved or dispersed repeats, and that the homogeneity of repeat arrays is affected by the number and the length of repeats, as well as by the presence of putative regulatory elements. We found that both tandem repeat organization and the presence of a TATA motif are significantly correlated with increased sequence similarity among homologous IGS repeats. We also observed that some repeat types are only found in a single species, while others appear to have persisted for >100 MY, with evidence for homologous repeat types in sister species. Taken together, these data suggest that both drift and natural selection influence repeat evolution within the IGS.

TATA binding protein binds to single stranded DNA: A new model for eukaryotic transcription

AbstractAlthough the players mediating the initiation of eukaryotic transcription appear to be known, the mechanism is not. In this paper we report findings on the interaction between the TATA box binding protein (TBP) and DNA, using a novel optical technology, that point to a potential plausible physical mechanism, and at the same time tie together a number of otherwise disparate observations. TBP plays a central and ubiquitous role in initiating eukaryotic transcription, binding to promoters with and without the canonical 5’ TATA(A/T)A(A/T)(G/A) 3’ motif1. It also tends to bind to double-stranded DNA promiscuously, and though affinity is lower, the number of binding opportunities is greater. TBP-DNA complexes have been characterized structurally2,3 and thermodynamically4,5, the standard model representing the TBP double-helix complex as the biologically significant entity. Here we show, using a label-free detection method termed Spectral Reflectance Imaging Biosensor6 (SRIB) that TBP binds to single stranded (ss), as well as double stranded (ds), oligonucleotides with the TATA motif, and binds the single-stranded form preferentially. Moreover, TBP binds preferentially to single stranded pyrimidine-rich sequences over purine-rich sequences, which is similar to the Single-Stranded DNA-binding protein (SSB) binding pattern. These observations suggest a new model for eukaryotic transcription in which TBP destabilizes the double helix, thereby initiating the open complex bubble necessary for transcription. Furthermore, the role of TBP as a multifunctional protein is discussed.

TATA binding protein binds to single stranded DNA: A new model for eukaryotic transcription

Although the players mediating the initiation of eukaryotic transcription appear to be known, the mechanism is not. In this paper we report findings on the interaction between the TATA box binding protein (TBP) and DNA, using a novel optical technology, that point to a potential plausible physical mechanism, and at the same time tie together a number of otherwise disparate observations. TBP plays a central and ubiquitous role in initiating eukaryotic transcription, binding to promoters with and without the canonical 5’ TATA(A/T)A(A/T)(G/A) 3’ motif1. It also tends to bind to double-stranded DNA promiscuously, and though affinity is lower, the number of binding opportunities is greater. TBP-DNA complexes have been characterized structurally2,3 and thermodynamically4,5, the standard model representing the TBP double-helix complex as the biologically significant entity. Here we show, using a label-free detection method termed Spectral Reflectance Imaging Biosensor6 (SRIB) that TBP binds to single stranded (ss), as well as double stranded (ds), oligonucleotides with the TATA motif, and binds the single-stranded form preferentially. Moreover, TBP binds preferentially to single stranded pyrimidine-rich sequences over purine-rich sequences, which is similar to the Single-Stranded DNA-binding protein (SSB) binding pattern. These observations suggest a new model for eukaryotic transcription in which TBP destabilizes the double helix, thereby initiating the open complex bubble necessary for transcription. Furthermore, the role of TBP as a multifunctional protein is discussed.

The scaRNA2 is produced by an independent transcription unit and its processing is directed by the encoding region.

The C/D box scaRNA2 is predicted to guide specific 2′-O-methylation of U2 snRNA. In contrast to other SCARNA genes, SCARNA2 appears to be independently transcribed. By transient expression of SCARNA2-reporter gene constructs, we have demonstrated that this gene is transcribed by RNA polymerase II and that the promoter elements responsible for its transcription are contained within a 161 bp region upstream of the transcription start site. In mammals, we have identified four cross species conserved promoter elements, a TATA motif, an hStaf/ZNF143 binding site and two novel elements that are required for full promoter activity. Binding of the human hStaf/ZNF143 transcription factor to its target sequence is required for promoter activity, suggesting that hStaf/ZNF143 is a fundamental regulator of the SCARNA2 gene. We also showed that RNA polymerase II continues transcription past the 3′-end of the mature RNA, irrespective of the identity of the Pol II promoter. The 3′-end processing and accumulation are governed by the sole information contained in the scaRNA2 encoding region, the maturation occurring via a particular pathway incompatible with that of mRNA or snRNA production.