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Cheng Ming Chiang - One of the best experts on this subject based on the ideXlab platform.
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the general transcription machinery and general cofactors
Critical Reviews in Biochemistry and Molecular Biology, 2006Co-Authors: Mary C Thomas, Cheng Ming ChiangAbstract:ABSTRACTIn eukaryotes, the core Promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or Downstream Promoter Element (DPE) found in most core Promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this Promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived ...
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The general transcription machinery and general cofactors.
Critical Reviews in Biochemistry and Molecular Biology, 2006Co-Authors: Mary C Thomas, Cheng Ming ChiangAbstract:ABSTRACTIn eukaryotes, the core Promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or Downstream Promoter Element (DPE) found in most core Promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this Promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived ...
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Sp1 and AP2 regulate but do not constitute TATA‐less human TAFII55 core Promoter activity
Nucleic acids research, 2002Co-Authors: Tianyuan Zhou, Cheng Ming ChiangAbstract:Human TAF(II)55 (hTAF(II)55), a component of the general transcription factor TFIID, is the only general transcription factor encoded by an intronless gene identified thus far. Analysis of the TATA-less hTAF(II)55 Promoter-proximal sequence reveals putative binding sites for STAT-1, MEF2, E2F, Sp1, AP2, AREB6 and E47. Using chromatin immunoprecipitation, DNase I footprinting and electrophoretic mobility shift assays, we demonstrate that Sp1 and AP2 can bind simultaneously to juxtaposed Sp1- and AP2-binding sites in the hTAF(II)55 Promoter-proximal region and functionally modulate hTAF(II)55 Promoter activity, as evidenced by reporter gene assays performed in transiently transfected human C-33A and insect SL2 cell lines. Interestingly, removal of all the Promoter-proximal Sp1-binding sites does not impair the function of the hTAF(II)55 core Promoter. Moreover, a 52-bp DNA fragment containing only the hTAF(II)55 initiator (Inr) and Downstream Promoter Element (DPE) is able to support Gal4-VP16-mediated activation in vivo and in vitro. Our data suggest that Sp1, although it plays an enhancing role in hTAF(II)55 gene expression, is not essential for hTAF(II)55 core Promoter activity. Interestingly, mutations introduced at the Inr and DPE differentially affect the selection of transcription start sites, suggesting that these two core Promoter Elements play a non-redundant role in the function of TATA-less Promoters.
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The intronless and TATA-less human TAF(II)55 gene contains a functional initiator and a Downstream Promoter Element.
The Journal of biological chemistry, 2001Co-Authors: Tianyuan Zhou, Cheng Ming ChiangAbstract:Human TAF(II)55 (hTAF(II)55) is a component of the multisubunit general transcription factor TFIID and has been shown to mediate the functions of many transcriptional activators via direct protein-protein interactions. To uncover the regulatory properties of the general transcription machinery, we have isolated the hTAF(II)55 gene and dissected the regulatory Elements and the core Promoter responsible for hTAF(II)55 gene expression. Surprisingly, the hTAF(II)55 gene has a single uninterrupted open reading frame and is the only intronless general transcription factor identified so far. Its expression is driven by a TATA-less Promoter that contains a functional initiator and a Downstream Promoter Element, as illustrated by both transfection assays and mutational analyses. Moreover, this core Promoter can mediate the activity of a transcriptional activator that is artificially recruited to the Promoter in a heterologous context. Interestingly, in the Promoter-proximal region there are multiple Sp1-binding sites juxtaposed to a single AP2-binding site, indicating that Sp1 and AP2 may regulate the core Promoter activity of the hTAF(II)55 gene. These findings indicate that a combinatorial regulation of a general transcription factor-encoding gene can be conferred by both ubiquitous and cell type-specific transcriptional regulators.
James T. Kadonaga - One of the best experts on this subject based on the ideXlab platform.
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Rational design of a super core Promoter that enhances gene expression
Nature Methods, 2006Co-Authors: Tamar Juven-gershon, Susan Cheng, James T. KadonagaAbstract:Transcription is a critical component in the expression of genes. Here we describe the design and analysis of a potent core Promoter, termed super core Promoter 1 (SCP1), which directs high amounts of transcription by RNA polymerase II in metazoans. SCP1 contains four core Promoter motifs—the TATA box, initiator (Inr), motif ten Element (MTE) and Downstream Promoter Element (DPE)—in a single Promoter, and is distinctly stronger than the cytomegalovirus (CMV) IE1 and adenovirus major late (AdML) core Promoters both in vitro and in vivo . Each of the four core Promoter motifs is needed for full SCP1 activity. SCP1 is bound efficiently by TFIID and exhibits a high propensity to form productive transcription complexes. SCP1 and related super core Promoters (SCPs) with multiple core Promoter motifs will be useful for the biophysical analysis of TFIID binding to DNA, the biochemical investigation of the transcription process and the enhancement of gene expression in cells.
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Enhancer-Promoter specificity mediated by DPE or TATA core Promoter motifs.
Genes & development, 2001Co-Authors: Jennifer E.f. Butler, James T. KadonagaAbstract:To investigate the basis for enhancer-Promoter specificity, we compared the ability of enhancers to activate transcription in vivo from core Promoters containing either Downstream Promoter Element (DPE) or TATA box motifs. To eliminate position effects, we generated and analyzed pairs of sister Drosophila lines that contain a DPE- or TATA-dependent reporter gene at precisely the same genomic position relative to each enhancer. These studies revealed transcriptional enhancers that are specific for Promoters that contain either DPE or TATA box Elements. Thus, the core Promoter not only mediates the initiation of transcription, but also functions as a regulatory Element.
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A Basal Transcription Factor That Activates or Represses Transcription
Science (New York N.Y.), 2000Co-Authors: Patricia J. Willy, Ryuji Kobayashi, James T. KadonagaAbstract:We have identified an activity that is required for transcription of Downstream Promoter Element (DPE)-containing core Promoters in vitro. The purified factor was found to be the Drosophila homolog of the transcriptional repressor known as NC2 or Dr1-Drap1. Purified recombinant dNC2 activates DPE-driven Promoters and represses TATA-driven Promoters. A mutant version of dNC2 can activate DPE Promoters but is unable to repress TATA Promoters. Thus, the activation and repression functions are distinct. These studies reveal that NC2 (Dr1-Drap1) is a bifunctional basal transcription factor that differentially regulates gene transcription through DPE or TATA box motifs.
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The Downstream Promoter Element DPE Appears To Be as Widely Used as the TATA Box in Drosophila Core Promoters
Molecular and cellular biology, 2000Co-Authors: Alan K. Kutach, James T. KadonagaAbstract:The Downstream Promoter Element (DPE) functions cooperatively with the initiator (Inr) for the binding of TFIID in the transcription of core Promoters in the absence of a TATA box. We examined the properties of sequences that can function as a DPE as well as the range of Promoters that use the DPE as a core Promoter Element. By using an in vitro transcription assay, we identified 17 new DPE-dependent Promoters and found that all possessed identical spacing between the Inr and DPE. Moreover, mutational analysis indicated that the insertion or deletion of a single nucleotide between the Inr and DPE causes a reduction in transcriptional activity and TFIID binding. To explore the range of sequences that can function as a DPE, we constructed and analyzed randomized Promoter libraries. These experiments yielded the DPE functional range set, which represents sequences that contribute to or are compatible with DPE function. We then analyzed the DPE functional range set in conjunction with a Drosophila core Promoter database that we compiled from 205 Promoters with accurately mapped start sites. Somewhat surprisingly, the DPE sequence motif is as common as the TATA box in Drosophila Promoters. There is, in addition, a striking adherence of Inr sequences to the Inr consensus in DPE-containing Promoters relative to DPE-less Promoters. Furthermore, statistical and biochemical analyses indicated that a G nucleotide between the Inr and DPE contributes to transcription from DPE-containing Promoters. Thus, these data reveal that the DPE exhibits a strict spacing requirement yet some sequence flexibility and appears to be as widely used as the TATA box in Drosophila.
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The Downstream core Promoter Element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila.
Genes & development, 1997Co-Authors: Thomas W. Burke, James T. KadonagaAbstract:Transcription is centrally involved in an array of biological processes, which include growth, development, and response to external stimuli. In eukaryotes, protein-coding genes are transcribed by the RNA polymerase II transcriptional machinery, which comprises RNA polymerase II and other factors that are required for basal and regulated transcription. Transcription by RNA polymerase II is directed by cis-acting DNA sequences that typically consist of a core Promoter along with regulatory Elements, such as enhancers, that contain binding sites for sequence-specific transcriptional activator and/or repressor proteins. Thus, the study of both the trans-acting protein factors and the cis-acting DNA Elements is necessary to gain a better understanding of the fundamental mechanisms by which genes are transcribed (for recent reviews, see Bjorkland and Kim 1996; Burley and Roeder 1996; Orphanides et al. 1996; Roeder 1996; Verrijzer and Tjian 1996; Ptashne and Gann 1997; Sauer and Tjian 1997; Smale 1997; Tansey and Herr 1997). The key DNA Element that is essential for transcription by RNA polymerase II is the core Promoter—the DNA sequences, which encompass the transcription start site (within about −40 to +40 relative to the +1 start site) and are sufficient to direct the accurate initiation of transcription. Two important core Promoter motifs are the TATA box and the initiator (Inr) (Fig. (Fig.1).1). The TATA box is an A/T-rich sequence that is located ∼25–30 nucleotides upstream of the RNA start site of many, but not all, Promoters. It is recognized by the TATA box-binding polypeptide (TBP), which is a component of the multisubunit TFIID complex. The Inr encompasses the RNA start site, and like the TATA box, it is also present in many, but not all, core Promoters (Smale and Baltimore 1989; Smale 1994, 1997). Inr Elements have been characterized in various TATA-less and TATA-containing Promoters, and the Inr consensus sequence is Py-Py-A+1-N-T/A-Py-Py (where A+1 is the transcription start site) in mammalian genes (Smale and Baltimore 1989; Bucher 1990; Javahery et al. 1994) and T-C-A+1-G/T-T-T/C in Drosophila genes (Hultmark et al. 1986; Purnell et al. 1994; Arkhipova 1995). Figure 1 The TATA box, Inr, and DPE are core Promoter Elements. The consensus sequences and locations of the TATA box, Inr, and DPE motifs are indicated. The TATA box and DPE appear to be functionally redundant, and Promoters generally do not contain ... Many Promoters contain functionally important sequences that are Downstream of the transcription start site. Such Downstream Promoter sequences have been found in TATA-containing Promoters (see, e.g., Lewis and Manley 1985; Nakatani et al. 1990; Lee et al. 1992; Emanuel and Gilmour 1993; Purnell and Gilmour 1993), as well as in TATA-less Promoters (see, e.g., Biggin and Tjian 1988; Perkins et al. 1988; Soeller et al. 1988; Smale and Baltimore 1989; Jarrell and Meselson 1991; Contursi et al. 1995; Minchiotti et al. 1997). It appears that many of these Downstream Promoter sequences are involved in basal transcription, but it is also important to consider that some Downstream Promoter sequences might be binding sites for sequence-specific transcriptional activators. In the analysis of the interaction of purified TFIID with TATA-less Promoters, a conserved Downstream core Promoter Element was found to be required for the sequence-specific binding of TFIID to a subset of TATA-less Promoters (Burke and Kadonaga 1996). This motif was termed the Downstream Promoter Element (DPE). The DPE is a distinct 7-nucleotide Element that is located at about +30 (typically, from +28 to +34) relative to the transcription start site (Fig. (Fig.1).1). It is present in many, but not all, Promoters and is bound by TFIID but not by TBP. In addition, nearly all of the Promoters that have been found to contain DPE-like sequences are TATA-less Promoters. Modification of the DPE by clustered point mutagenesis was found to cause a decrease in the binding of purified TFIID to the Promoter as well as an 8- to 100-fold reduction in basal transcriptional activity in vitro. Mutational analysis of the Inr and DPE motifs revealed that the DPE acts in conjunction with the Inr to provide a binding site for TFIID in the absence of a TATA box. Interestingly, the loss of transcriptional activity upon disruption of the TATA box in a TATA-containing Promoter could be recovered by the insertion of a DPE at a Downstream position (+28 to +34) in the defective Promoter. Thus, the DPE appears to be a functionally important, conserved Downstream core Promoter Element. We are, however, still in the early stages of understanding the DPE. In this work we have investigated several fundamental questions regarding the role of the DPE in the transcription process. These experiments provide evidence for the function of TBP-associated factors (TAFs) in DPE-driven basal transcription and reveal that the DPE has many properties that are analogous to those of the TATA box.
Juan L. Ramos - One of the best experts on this subject based on the ideXlab platform.
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Hierarchical Binding of the TodT Response Regulator to Its Multiple Recognition Sites at the tod Pathway Operon Promoter
Journal of molecular biology, 2007Co-Authors: Jesús Lacal, Andreas Busch, María-eugenia Guazzaroni, Tino Krell, Juan L. RamosAbstract:Abstract The TodS and TodT proteins form a highly specific two-component regulatory system that controls the expression of genes involved in the degradation of toluene, benzene, and ethylbenzene via the toluene dioxygenase pathway. The catabolic genes of the toluene dioxygenase pathway are transcribed from a single Promoter called P todX once the response regulator TodT is phosphorylated by the TodS sensor kinase in response to pathway substrates. We show here that TodT is a monomer in solution and that it binds to three specific sites in the P todX Promoter, centered at − 57, − 85, and − 106 with respect to the transcription start site. The − 85 and − 106 sites are pseudopalindromic, whereas the − 57 site is half a palindrome. TodT binding to its target sites is sequential, as shown by electrophoresis mobility gel shift assays and footprinting. The binding affinity values of TodT, as determined by isothermal titration calorimetry, are 1.8 ± 0.2, 5 ± 0.4, and 6.3 ± 0.8 μM for the − 106, − 85, and − 57 sites, respectively, and the binding stoichiometry is one monomer per half-palindromic Element. Mutational analysis revealed that all three sites contribute to P todX strength, although the most relevant site is the distal one with respect to the − 10 extended Element of the Downstream Promoter Element. The C-TodT [C-terminal TodT fragment (amino acids 154–206)], a truncated variant of TodT that contains the C-terminal half of the protein bearing the DNA binding domain, binds in vitro to all three sites with affinity similar to that of the full-length protein. However, C-TodT, in contrast to the full-length regulator, does not activate in vitro transcription from P todX . We discuss the consequences of the organization of the binding sites on transcriptional control and propose that the N-terminal domain of TodT is necessary for appropriate interactions with other transcriptional Elements.
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The TodS-TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Jesús Lacal, Andreas Busch, María-eugenia Guazzaroni, Tino Krell, Juan L. RamosAbstract:The TodS and TodT proteins form a previously unrecognized and highly specific two-component regulatory system in which the TodS sensor protein contains two input domains, each of which are coupled to a histidine kinase domain. This system regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. In contrast to the narrow substrate range of this catabolic pathway, the TodS effector profile is broad. TodS has basal autophosphorylation activity in vitro, which is enhanced by the presence of effectors. Toluene binds to TodS with high affinity (Kd = 684 ± 13 nM) and 1:1 stoichiometry. The analysis of the truncated variants of TodS reveals that toluene binds to the N-terminal input domain (Kd = 2.3 ± 0.1 μM) but not to the C-terminal half. TodS transphosphorylates TodT, which binds to two highly similar DNA binding sites at base pairs −107 and −85 of the Promoter. Integration host factor (IHF) plays a crucial role in the activation process and binds between the upstream TodT boxes and the −10 hexamer region. In an IHF-deficient background, expression from the tod Promoter drops 8-fold. In vitro transcription assays confirmed the role determined in vivo for TodS, TodT, and IHF. A functional model is presented in which IHF favors the contact between the TodT activator, bound further upstream, and the α-subunit of RNA polymerase bound to the Downstream Promoter Element. Once these contacts are established, the tod operon is efficiently transcribed.
Danny Reinberg - One of the best experts on this subject based on the ideXlab platform.
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Functional Characterization of Core Promoter Elements: The Downstream Core Element is Recognized by TAF1
Molecular and Cellular Biology, 2005Co-Authors: Dong-hoon Lee, Danny Reinberg, Naum I. Gershenzon, Malavika Gupta, Ilya Ioshikhes, Brian A LewisAbstract:Downstream Elements are a newly appreciated class of core Promoter Elements of RNA polymerase II-transcribed genes. The Downstream core Element (DCE) was discovered in the human β-globin Promoter, and its sequence composition is distinct from that of the Downstream Promoter Element (DPE). We show here that the DCE is a bona fide core Promoter Element present in a large number of Promoters and with high incidence in Promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse Promoters, supporting its functional relevance in a variety of Promoter contexts. The DCE consists of three subElements, and DCE function is recapitulated in a TFIID-dependent manner. SubElement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAFII250 interacts with the DCE subElement DNA in a sequence-dependent manner. These data show that Downstream Elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core Promoter recognition complex.
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functional characterization of core Promoter Elements dpe specific transcription requires the protein kinase ck2 and the pc4 coactivator
Molecular Cell, 2005Co-Authors: Brian A Lewis, Robert J Sims, William S Lane, Danny ReinbergAbstract:Downstream core Promoter Elements are an expanding class of regulatory sequences that add considerable diversity to the Promoter architecture of RNA polymerase II-transcribed genes. We set out to determine the factors necessary for Downstream Promoter Element (DPE)-dependent transcription and find that, against expectations, TFIID and the GTFs are not sufficient. Instead, the protein kinase CK2 and the coactivator PC4 establish DPE-specific transcription in an in vitro transcription system containing TFIID, Mediator, and the GTFs. Chromatin immunoprecipitation analyses using the DPE-dependent IRF-1 and TAF7 Promoters demonstrated that CK2, and PC4 are present on these Promoters in vivo. In contrast, neither PC4 nor CK2 were detected on the TAF1-dependent cyclin D Promoter, which contains a DCE type of Downstream Element. Our findings also demonstrate that CK2 activity alters TFIID-dependent recognition of DCE sequences. These data establish that CK2 acts as a switch, converting the transcriptional machinery from functioning on one type of Downstream Element to another.
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New core Promoter Element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB
Genes & development, 1998Co-Authors: Thierry Lagrange, Danny Reinberg, Achillefs N. Kapanidis, Hong Tang, Richard H. EbrightAbstract:A sequence Element located immediately upstream of the TATA Element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence Element is recognized directly by the transcription factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core Promoter Element, the IIB recognition Element (BRE), in addition to the TATA Element, the initiator Element, and the Downstream Promoter Element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID.
Sascha H Duttke - One of the best experts on this subject based on the ideXlab platform.
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rna polymerase iii accurately initiates transcription from rna polymerase ii Promoters in vitro
Journal of Biological Chemistry, 2014Co-Authors: Sascha H DuttkeAbstract:Abstract In eukaryotes, there are three major RNA polymerases (Pol) in the nucleus, which are commonly described as transcribing non-overlapping subsets of genes. Structural studies have highlighted a conserved core shared among all three transcription systems. Initiation of human Pol III from TATA box-containing Pol II Promoters under conditions with impaired Pol II transcription activity have been described previously. RNA polymerase III and Pol II were found to co-localize at the Promoters of the c-myc gene and the RPPH1 sRNA in vivo. Here, I report that Pol III can, like Pol II, initiate transcription from most tested Pol II core Promoters when assayed with crude human nuclear extracts (HSK, SNF, or Dignam). Both polymerases often initiate from the same transcription start site, and depend on a TATA box or AT-rich region but not the Downstream Promoter Element (DPE) or the motif ten Element (MTE). Moderate (∼2-fold) changes in the ratio of DNA template to nuclear extract were sufficient to change Pol II-mediated transcription to a mixture of Pol II- and Pol III-, or to a solely Pol III-dependent initiation of transcription from Pol II Promoters. Polymerase specificity is thus not fixed but a variable that depends on the properties of the Promoter and the transcription conditions. These findings provide functional evidence for a close similarity between the Pol II and Pol III transcription complexes, and additionally explain previous controversies in the literature.