Language：English   Publishing type：Research paper (scientific journal)   Publisher：Genetics Society of Japan  

DOI： 10.1266/ggs.19-00001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

In Saccharomyces cerevisiae, core promoters of class II genes contain a TATA element, either a TATA box (TATA[A/T]A[A/T][A/G]) or TATA-like element (1 or 2 bp mismatched version of the TATA box). The TATA element directs the assembly of the preinitiation complex (PIC) to ensure accurate transcriptional initiation. It has been proposed the PIC is assembled by two distinct pathways in which TBP is delivered by TFIID or SAGA, leading to the widely accepted model that these complexes mediate transcription mainly from TATA-like element-or TATA box-containing promoters, respectively. Although both complexes are involved in transcription of nearly all class II genes, it remains unclear how efficiently SAGA mediates transcription from TATA-like element-containing promoters independently of TFIID. We found that transcription from the TATA box-containing AGP1 promoter was greatly stimulated in a Spt3p-dependent manner after inactivation of Taf1p/TFIID. Thus, this promoter provides a novel experimental system in which to evaluate SAGA-mediated transcription from TATA-like element(s). We quantitatively measured transcription from various TATA-like elements in the Taf1p-dependent CYC1 promoter and Taf1p-independent AGP1 promoter. The results revealed that SAGA could mediate transcription from at least some TATA-like elements independently of Taf1p/TFIID, and that Taf1p-dependence or -independence is highly robust with respect to variation of the TATA sequence. Furthermore, chimeric promoter mapping revealed that Taf1p-dependence or independence was conferred by the upstream activating sequence (UAS), whereas Spt3p-dependent transcriptional stimulation after inactivation of Taf1p/TFIID was specific to the AGP1 promoter and dependent on core promoter regions other than the TATA box. These results suggest that TFIID and/or SAGA are regulated in two steps: the UAS first specifies TFIID or SAGA as the predominant factor on a given promoter, and then the core promoter structure guides the pertinent factor to conduct transcription in an appropriate manner.

DOI： 10.1371/journal.pone.0188435

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Hmo1, a member of HMGB family proteins in Saccharomyces cerevisiae, binds to and regulates the transcription of genes encoding ribosomal RNA and ribosomal proteins. The functional motifs of Hmo1 include two HMG-like motifs, box A and box B, and a C-terminal tail. To elucidate the molecular roles of the HMG-like boxes in DNA binding in vivo, we analyzed the DNA-binding activity of various Hmo1 mutants using ChIP or reporter assays that enabled us to conveniently detect Hmo1 binding to the promoter of RPS5, a major target gene of Hmo1. Our mutational analyses showed that box B is a bona fide DNA-binding motif and that it also plays other important roles in cell growth. However, box A, especially its first alpha-helix, contributes to DNA binding of Hmo1 by inducing self-assembly of Hmo1. Intriguingly, box A mediated formation of oligomers of more than two proteins on DNA in vivo. Furthermore, duplication of the box B partially alleviates the requirement for box A. These findings suggest that the principal role of box A is to assemble multiple box B in the appropriate orientation, thereby stabilizing the binding of Hmo1 to DNA and nucleating specific chromosomal architecture on its target genes.

DOI： 10.1111/gtc.12449

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

In Saccharomyces cerevisiae, the core promoters of class II genes contain either TATA or TATA-like elements to direct accurate transcriptional initiation. Genome-wide analyses show that the consensus sequence of the TATA element is TATAWAWR (8 bp), whereas TATA-like elements carry one or two mismatches to this consensus. The fact that several functionally distinct TATA sequences have been identified indicates that these elements may function, at least to some extent, in a gene-specific manner. The purpose of the present study was to identify functional TATA sequences enriched in one particular core promoter and compare them with the TATA or TATA-like elements that serve as the pre-initiation complex (PIC) assembly sites on the yeast genome. For this purpose, we conducted a randomized screen of the TATA element in the CYC1 promoter by using a novel reporter assay system and identified several hundreds of unique sequences that were tentatively classified into nine groups. The results indicated that the 7 bp TATA element (i.e., TATAWAD) and several sets of TATA-like sequences are preferred specifically by this promoter. Furthermore, we find that the most frequently isolated TATA-like sequence, i.e., TATTTAAA, is actually utilized as a functional core promoter element for the endogenous genes, e.g., ADE5,7 and ADE6. Collectively, these results indicate that the sequence requirements for the functional TATA or TATA-like elements in one particular core promoter are not as stringent. However, the variation of these sequences differs significantly from that of the PIC assembly sites on the genome, presumably depending on promoter structures and reflecting the gene-specific function of these sequences.

DOI： 10.1371/journal.pone.0129357

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0124655

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Hmo1, a member of the high mobility group B family proteins in Saccharomyces cerevisiae, associates with the promoters of ribosomal protein genes (RPGs) to direct accurate transcriptional initiation. Here, to identify factors involved in the binding of Hmo1 to its targets and the mechanism of Hmo1-dependent transcriptional initiation, we developed a novel reporter system using the promoter of the RPG RPS5. A genetic screen did not identify any factors that influence Hmo1 binding, but did identify a number of mutations in Hmo1 that impair its DNA binding activity in vivo and in vitro. These results suggest that Hmo1 binds to its target promoters autonomously without any aid of additional factors. Furthermore, characterization of Hmo1 mutants showed that the box A domain plays a pivotal role in DNA binding and may be required for the recognition of structural properties of target promoters that occur in native chromatin.

DOI： 10.1080/09168451.2014.978258

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP PUBLISHING LTD  

Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (similar to 50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1-3MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulations to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain. Due to this property, once a large complex binds its target site via gene-specific factors it can act as a 'buoy' to keep the target region on the surface of the condensed domain and maintain transcriptional competency. This size-dependent specialization of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination.

DOI： 10.1088/0953-8984/27/6/064116

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

DOI： 10.1038/nsmb.2611

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the beta subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the beta subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF Al domain and GPIb alpha indicated that Asp62, Arg115, and Lys117 of the beta subunit are located near Arg218 and Asp222 of GPIb alpha, respectively, and that Asp beta 70 is in proximity to Gln1391 of the Al domain. Our results indicate that these charged amino acid residues in the beta subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

DOI： 10.1021/bi300442c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Ig gene hypermutation is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. One of the unsolved mysteries is how AID-triggered U:G lesions result in efficient induction of mutations at non-damaged A/T bases in the V(H) genes of germinal center (GC) B cells. Genetic and biochemical evidence suggests that components of the mismatch repair pathway and the low fidelity DNA polymerase eta are required for the induction of A/T mutations. However, mismatch repair proficient NIH3T3 cells are unable to generate a high frequency of A/T mutations, even after DNA polymerase eta overexpression, suggesting that additional mechanisms are involved. Since GC B cells undergo enormous expansion while undergoing hypermutation, we hypothesized that rapid cell division might play a role in the induction of A/T mutations. To test this hypothesis, we utilized an efficient in vitro mutagenesis system, which closely mirrors physiological Ig gene hypermutation, in the human GC-like B cell line Ramos. Ramos cells transduced with AID-IRES-GFP retrovirus were cultured for 10 days in medium supplemented with 20% or 2% fetal bovine serum (FBS) to allow rapid and slow proliferation, respectively. Analysis of the V(H) gene mutations revealed that A/T mutations were significantly reduced in 2% FBS compared with 20% FBS, with transitions more affected than transversions. These results demonstrate that rapid cell division contributes to efficient induction of A/T mutations and suggest that the rate of DNA replication has a profound effect on the processing of AID-triggered U:G lesions. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.molimm.2011.06.218

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC AM  

In diploid Saccharomyces cerevisiae cells, bud-site selection is determined by two cortical landmarks, Bud8p and Bud9p, at the distal and proximal poles, respectively. Their localizations depend on the multigenerational proteins Rax1p/Rax2p. Many genes involved in bud-site selection were identified previously by genome-wide screening of deletion mutants, which identified BUD32 that causes a random budding in diploid cells. Bud32p is an atypical kinase involved in a signaling cascade of Sch9p kinase, the yeast homolog of Akt/PKB, and a component of the EKC/KEOPS (endopeptidase-like, kinase, chromatin-associated/kinase, putative endopeptidase, and other proteins of small size) complex that functions in telomere maintenance and transcriptional regulation. However, its role in bipolar budding has remained unclear. In this report, we show that the Sch9p kinase cascade does not affect bipolar budding but that the EKC/KEOPS complex regulates the localization of Bud9p. The kinase activity of Bud32p, which is essential for the functions of the EKC/KEOPS complex but is not necessary for the Sch9p signaling cascade, is required for bipolar bud-site selection. BUD9 is necessary for random budding in each deletion mutant of EKC/KEOPS components, and RAX2 is genetically upstream of EKC/KEOPS genes for the regulation of bipolar budding. The asymmetric localization of Bud9p was dependent on the complex, but Bud8p and Rax2p were not. We concluded that the EKC/KEOPS complex is specifically involved in the regulation of Bud9p localization downstream of Rax1p/Rax2p.

DOI： 10.1534/genetics.111.128231

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Saccharomyces cerevisiae Hmo1 binds to the promoters of similar to 70% of ribosomal protein genes (RPGs) at high occupancy, but is observed at lower occupancy on the remaining RPG promoters. In delta hmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift. Analyses of chimeric RPS5/RPL10 promoters revealed a region between the RPS5 upstream activating sequence (UAS) and core promoter, termed the intervening region (IVR), responsible for strong Hmo1 binding and an upstream TSS shift in delta hmo1 cells. Chromatin immunoprecipitation analyses showed that the RPS5-IVR resides within a nucleosome-free region and that pre-initiation complex (PIC) assembly occurs at a site between the IVR and a nucleosome overlapping the TSS (+1 nucleosome). The PIC assembly site was shifted upstream in delta hmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s). This novel mechanism ensures accurate transcriptional initiation by delineating the 5'- and 3'-boundaries of the PIC assembly zone.

DOI： 10.1093/nar/gkq1334

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In eukaryotes, protein-coding genes are transcribed by RNA polymerase II (pol II) together with general transcription factors (GTFs). TFIID, the largest GTF composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a critical role in transcription from TATA-less promoters. In metazoans, several core promoter elements other than the TATA element are thought to be recognition sites for TFIID. However, it is unclear whether functionally homologous elements also exist in TATA-less promoters in Saccharomyces cerevisiae. Here, we identify the cis-elements required to support normal levels of transcription and accurate initiation from sites within the TATA-less and TFIID-dependent RPS5 core promoter. Systematic mutational analyses show that multiple AT-rich sequences are required for these activities and appear to function as recognition sites for TFIID. A single copy of these sequences can support accurate initiation from the endogenous promoter, indicating that they carry highly redundant functions. These results show a novel architecture of yeast TATA-less promoters and support a model in which pol II scans DNA downstream from a recruited site, while searching for appropriate initiation site(s).

DOI： 10.1093/nar/gkq741

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In Saccharomyces cerevisiae, TFIID, which is composed of TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs), mediates the transcription of most class II genes. Previous studies have shown that CLN2 expression was significantly reduced by taf1-ts2, but not by taf1-N568 Delta, although both mutations display similar temperature-sensitive growth phenotypes and transcriptional defects in other genes. Here, we show that the reduced expression of CLN2 is not because of differences in taf1 alleles in the previous experiments but because of allelic differences at the SSD1 locus in the host strains. Specifically, ssd1-d reduces CLN2 expression when combined with taf1. Ssd1p expressed from SSD1-V, but not from ssd1-d, stabilizes a subpopulation of CLN2 mRNA in wild-type and taf1-N568 Delta strains and facilitates the continuous transcription of CLN2 after heat shock in the taf1-N568 Delta strain. Reporter assays show that both activities appear to depend on the 5'-untranslated region of CLN2 mRNA and that Ssd1p binds to this region via its amino- and carboxy-terminal domains. Based on these observations, we propose a model for the action of Ssd1p and discuss its biologic role.

DOI： 10.1111/j.1365-2443.2010.01452.x

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Language：English   Publishing type：Research paper (scientific journal)  

In Saccharomyces cerevisiae, TFIID, which is composed of TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs), mediates the transcription of most class II genes. Previous studies have shown that CLN2 expression was significantly reduced by taf1-ts2, but not by taf1-N568Δ, although both mutations display similar temperature-sensitive growth phenotypes and transcriptional defects in other genes. Here, we show that the reduced expression of CLN2 is not because of differences in taf1 alleles in the previous experiments but because of allelic differences at the SSD1 locus in the host strains. Specifically, ssd1-d reduces CLN2 expression when combined with taf1. Ssd1p expressed from SSD1-V, but not from ssd1-d, stabilizes a subpopulation of CLN2 mRNA in wild-type and taf1-N568Δ strains and facilitates the continuous transcription of CLN2 after heat shock in the taf1-N568Δ strain. Reporter assays show that both activities appear to depend on the 5'-untranslated region of CLN2 mRNA and that Ssd1p binds to this region via its amino- and carboxy-terminal domains. Based on these observations, we propose a model for the action of Ssd1p and discuss its biologic role. © 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2443.2010.01452.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In Saccharomyces cerevisiae, TFIID and SAGA principally mediate transcription of constitutive housekeeping genes and stress-inducible genes, respectively, by delivering TBP to the core promoter. Both are multi-protein complexes composed of 15 and 20 subunits, respectively, five of which are common and which may constitute a core sub-module in each complex. Although genome-wide gene expression studies have been conducted extensively in several TFIID and/or SAGA mutants, there are only a limited number of studies investigating genome-wide localization of the components of these two complexes. Specifically, there are no previous reports on localization of a complete set of Tafs and the effects of taf mutations on localization. Here, we examine the localization profiles of a complete set of Tafs, Gcn5, Bur6/Ncb2, Sua7, Tfa2, Tfg1, Tfb3 and Rpb1, on chromosomes III, IV and V by chromatin immunoprecipitation (ChIP)-chip analysis in wild-type and taf1-T657K mutant strains. In addition, we conducted conventional and sequential ChIP analysis of several ribosomal protein genes (RPGs) and non-RPGs. Intriguingly, the results revealed a novel relationship between TFIIB and NC2, simultaneous co-localization of SAGA and TFIID on RPG promoters, specific effects of taf1 mutation on Taf2 occupancy, and an indirect evidence for the existence of different TFIID conformations.

DOI： 10.1093/nar/gkp1172

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The micromeres of sea urchin embryos have two functions: to promote the autonomous differentiation of skeletogenic cells and to induce endomesodermal tissues. Micromere specification is controlled by a double-repression gate consisting of two repressors, Pmar1 and HesC. Micro1/pmar1 encodes a transcriptional repressor with a paired-type N-terminal homeodomain and two C-terminal serine-rich repeats, each of which includes a sequence similar to engrailed homology region 1, which interacts with the co-repressor Groucho. To understand the molecular mechanisms of the double-repression gate, we examined the correlation between the structure and function of micro1. Phenotypic and gene expression pattern analyses of embryos injected with mutated micro1 mRNA revealed that micro1 consists of five functional domain and motifs; namely, a DNA-binding homeodomain, a nuclear localization signal in the C-terminal flanking region of the homeodomain, and two eh1-like motifs plus a short C-terminal stretch that together mediate transcriptional repression. Our data suggest that micro1 represses target genes, including hesC, via two redundant means: its eh1-like and C-terminal motifs. The C-terminal motif requires unidentified sequences for micro1 function; a micro1 mutant with the motif but lacking the unidentified sequences failed to trigger the double-repression gate for early micromere regulatory genes, except for delta, though it did repress hesC. Our results suggest that the spatial regulation of primary mesenchyme cell specification genes, including tbr, alx1, and ets1, may be different from that of delta. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2009.06.1083

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Mediator is one of the most important co-activators that function in eukaryotic transcriptional regulation. In Saccharomyces cerevisiae, Mediator is comprised of 25 subunits belonging to four structurally distinct modules: Head, Middle, Tail, and Cyc-C. Although each module plays a critical role in the regulation of a distinct set of genes, the precise molecular mechanisms remain unclear. To gain new insight into the role of the less-characterized Middle module, we analyzed the function of Med9 by constructing a set of mutants and subjecting them to a range of in vivo and in vitro assays. Our results demonstrate that Med9 has two functional domains. The species-specific amino-terminal half (aa 1-63) plays a regulatory role in transcriptional regulation in vivo and in vitro. In contrast, the well-conserved carboxy-terminal half (aa 64-149) has a more fundamental function involved in direct binding to the amino-terminal portions of Med4 and Med7 and the assembly of Med9 into the Middle module. Importantly, activator-dependent recruitment of TBP and Taf11 to the promoter is differentially affected in med9 extracts and in extracts lacking Mediator. Add-back experiments indicate that some unidentified factor(s) in med9 extracts may impact the binding of TFIID to the promoter.

DOI： 10.1111/j.1365-2443.2008.01250.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Heat shock factor 2 (HSF2) is a member of a vertebrate transcription factor family for genes of heat shock proteins and is involved in the regulation of development and cellular differentiation. The DNA binding property of HSF2 is modulated by the post-translational modification of a specific lysine residue in its DNA binding domain by small ubiquitin-like modifier (SUMO), but the consequences of SUMOylation and its underlying molecular mechanism remain unclear. Here we show the inhibitory effect of SUMOylation on the interaction between HSF2 and DNA based on biochemical analysis using isolated recombinant HSF2. NMR study of the SUMOylated DNA binding domain of HSF2 indicates that the SUMO moiety is flexible with respect to the DNA binding domain and has neither a noncovalent interface with nor a structural effect on the domain. Combined with data from double electron-electron resonance and paramagnetic NMR relaxation enhancement experiments, these results suggest that SUMO attachment negatively modulates the formation of the protein-DNA complex through a randomly distributed steric interference.

DOI： 10.1074/jbc.M806392200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Saccharomyces cerevisiae HMO1, a high mobility group B ( HMGB) protein, associates with the rRNA locus and with the promoters of many ribosomal protein genes ( RPGs). Here, the Sos recruitment system was used to show that HMO1 interacts with TBP and the N-terminal domain ( TAND) of TAF1, which are integral components of TFIID. Biochemical studies revealed that HMO1 copurifies with TFIID and directly interacts with TBP but not with TAND. Deletion of HMO1 (Delta hmo1) causes a severe cold-sensitive growth defect and decreases transcription of some TAND-dependent genes. Dhmo1 also affects TFIID occupancy at some RPG promoters in a promoter-specific manner. Interestingly, over-expression of HMO1 delays colony formation of taf1 mutants lacking TAND (taf1 Delta TAND), but not of the wild-type strain, indicating a functional link between HMO1 and TAND. Furthermore, Delta hmo1 exhibits synthetic growth defects in some spt15 ( TBP) and toa1 (TFIIA) mutants while it rescues growth defects of some sua7 ( TFIIB) mutants. Importantly, Dhmo1 causes an upstream shift in transcriptional start sites of RPS5, RPS16A, RPL23B, RPL27B and RPL32, but not of RPS31, RPL10, TEF2 and ADH1, indicating that HMO1 may participate in start site selection of a subset of class II genes presumably via its interaction with TFIID.

DOI： 10.1093/nar/gkm1068

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.ab.2007.08.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

HMO1 is a high-mobility group B protein that plays a role in transcription of genes encoding rRNA and ribosomal proteins (RPGs) in Saccharomyces cerevisiae. This study uses genome-wide chromatin immunoprecipitation to study the roles of HMO1, FHL1, and RAP1 in transcription of these genes as well as other RNA polymerase II-transcribed genes in yeast. The results show that HMO1 associates with the 35S rRNA gene in an RNA polymerase I-dependent manner and that RPG promoters (138 in total) can be classified into several distinct groups based on HMO1 abundance at the promoter and the HMO1 dependence of FHL1 and/or RAP1 binding to the promoter. FHL1, a key regulator of RPGs, binds to most of the HMO1-enriched and transcriptionally HMO1-dependent RPG promoters in an HMO1-dependent manner, whereas it binds to HMO1-limited RPG promoters in an HMO1-independent manner, irrespective of whether they are transcribed in an HMO1-dependent manner. Reporter gene assays indicate that these functional properties are determined by the promoter sequence.

DOI： 10.1128/MCB.00876-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

General transcription factor TFIID is comprised of TATA-binding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10-37) and TAND2 (residues 46-71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6-71) was hindered by its poor solubility and stability in solution. Here we report a molecular engineering approach where the N terminus of TBP is fused to the C terminus of TAND12 via linkers of various lengths containing (GGGS)(n) sequence, (n = 1, 2, 3). The length of the linker within the TAND12-TBP fusion has a significant effect on solubility and stability (SAS). The construct with (GGGS)(3) linker produces the best quality single-quantum-coherence (HSQC) NMR spectrum with markedly improved SAS. In parallel to these observations, the TAND12-TBP fusion exhibits marked reduction of TBP function in binding to TAF1 as well as temperature sensitivity in in vivo yeast cell growth. Remarkably, the temperature sensitivity was proportional to the length of the linker in the fusions: the construct with (GGGS)(3) linker did not grow at 20 degrees C, while those with (GGGS)(1) and (GGGS)(2) linkers did. These results together indicate that the native interaction between TBP and TAND12 is well maintained in the TAND12-(GGGS)(3)-TBP fusion and that this fusion approach provides an excellent model system to investigate the structural detail of the TBP-TAF1 interaction.

DOI： 10.1074/jbc.M702988200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

There are some functional compatibilities between upstream and core promoter sequences for transcriptional activation in yeast, Drosophila and mammalian cells. Here we examined whether similar compatibilities exist in sea urchin embryos, and if so, whether they are dynamically regulated during early development. Two reporter plasmids, each containing a test promoter conjugated to either CFP or YFP, were concurrently introduced into embryos, and their expression patterns were studied by fluorescence microscopy. The upstream sequence of the Hemicentrotus pulcherrimus ( Hp) OtxE promoter drives the expression of its own core promoter and that of Strongylocentrotus purpuratus (Sp) Spec2a in different embryonic regions, especially at the late gastrula stage. Interestingly, when the four putative transcription factor binding sites of this upstream sequence were individually mutated, the resulting sequences directed different spatio-temporal expression from the same set of two core promoters, indicating that combinations of upstream factors may determine core promoter usage in sea urchin embryos. In addition, the insertion or deletion of consensus or nonconsensus TATA sequences changed the expression profile significantly, irrespective of whether the upstream sequence was intact or mutated. Thus, the TATA sequence may serve as a primary determinant for core promoter selection in these cells.

DOI： 10.1093/nar/gkm519

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Gene expression reporter systems, in which a promoter of interest is cloned upstream of a readily assayed reporter gene, have been developed and used extensively to study gene expression in prokaryotes and eukaryotes. Unfortunately, most of these systems cannot be used to assay gene expression in nonsuperficial tissues in living organisms. This study examines a novel reporter gene system based on the gene encoding Escherichia coli polyphosphate kinase (PPK), which can be used to monitor gene expression in mammalian cells. PPK catalyzes the synthesis of inorganic polyphosphate (polyP) from ATP and because mammalian cells do not contain detectable levels of polyP PPK activity can be measured in mammalian cells using (31)P-magnetic resonance spectroscopy or (31)P-magnetic resonance imaging. The ppk reporter gene system described here is noninvasive, does not require an exogenous substrate, and can potentially be used in internal tissues of living organisms.

DOI： 10.2144/000112319

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. N-15-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional H-1-N-15 correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein-protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.

DOI： 10.1007/s10858-006-9079-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In unicellular and multicellular eukaryotes, elaborate gene regulatory mechanisms facilitate a broad range of biological processes from cell division to morphological differentiation. In order to fully understand the gene regulatory networks involved in these biological processes, the spatial and temporal patterns of expression of many thousands of genes will need to be determined in real time in living organisms. Currently available techniques are not sufficient to achieve this goal; however, novel methods based on magnetic resonance (MR) imaging may be particularly useful for sensitive detection of gene expression in opaque tissues. This report describes a novel reporter gene system that monitors gene expression dynamically and quantitatively, in yeast cells, by measuring the accumulation of inorganic polyphosphate (polyP) using MR spectroscopy (MRS) or MR spectroscopic imaging (MRI). Because this system is completely non-invasive and does not require exogenous substrates, it is a powerful tool for studying gene expression in multicellular organisms, as well.

DOI： 10.1093/nar/gkl135

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S119_3

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DOI： 10.1007/s10858-005-1929-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/j.1356-9597.2004.00762.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

General transcription factor TRID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription. The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes. We have previously demonstrated that the Drosophila TAND region of TAF1 (residues 11-77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP. In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro. Here we present structural and functional studies of yeast TAND1 and TAND2 regions (residues 10-37, and 46-71, respectively). Our NMR data show that, in yeast, TAND1 contains two alpha-helices (residues 16-23, 30-36) and TAND2 forms a mini beta-sheet structure (residues 53-56, 61-64). These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively. Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex alpha-helix 2, overlaps with the mini beta-sheet region. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2004.04.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The general transcription factor TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID mediates the transcriptional activation of a subset of eukaryotic promoters. The N-terminal domain (TAND) of TAF1 protein (Taf1p) inhibits TBP by binding to its concave and convex surfaces. This study examines the role of the TAND in transcriptional regulation and tests whether the TAND is an autonomous regulator of TBP. The TAND binds to and regulates TBP function when it is fused to the amino or carboxy terminus of Taf1p, the amino or carboxy terminus of Taf5p, or the amino terminus of Tan11p. However, a carboxy-terminal fusion of the TAND and Taf11p is not compatible with several other TAF proteins, including Taf11p, in the TFIID complex. These results indicate that there is no or minimal geometric constraint on the ability of the TAND to function normally in transcriptional regulation as long as TFHD assembly is secured.

DOI： 10.1128/MCB.24.8.3089-3099.2004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

TFIID, a multiprotein complex composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), can directly recognize core promoter elements and mediate transcriptional activation. The TAF N-terminal domain (TAND) of TAF1 may play a significant role in these two principal TFIID functions by regulating the access of TBP to the TATA element. In yeast, TAND consists of two subdomains, TAND1 (10-37 amino acids (aa)) and TAND2 (46-71 aa), which interact with the concave and convex surfaces of TBP, respectively. Here we demonstrate that another region located on the C-terminal side of TAND2 (82-139 aa) can also bind to TBP and induce transcriptional activation when tethered to DNA as a GAL4 fusion protein. As these properties are the same as those of TAND1, we denoted this sequence as TAND3. Detailed mutational analyses revealed that three blocks of hydrophobic amino acid residues located within TAND3 are required not only for TBP binding and transcriptional activation but also for supporting cell growth and the efficient transcription of a subset of genes. We also show that the surface of TBP recognized by TAND3 is broader than that recognized by TAND1, although these regions overlap partially. Supporting these observations is that TAND1 can be at least partly functionally substituted by TAND3.

DOI： 10.1074/jbc.M306886200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2003.09.050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

DOI： 10.1074/jbc.m213220200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The general transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), is important for both basal and regulated transcription by RNA polymerase II. Although it is well known that the TAF N-terminal domain (TAND) at the amino-terminus of the TAF1 protein binds to TBP and thereby inhibits TBP function in vitro, the physiological role of this domain remains obscure. In our previous study, we screened for mutations that cause lethality when co-expressed with the TAF1 gene lacking TAND (taf1-DeltaTAND) and identified two DeltaTAND synthetic lethal (nsl) mutations as those in the SPT15 gene encoding TBP. In this study we isolated another nsl mutation in the same screen and identified it to be a mutation in the histone fold domain (HFD) of the TAF12 gene. Several other HFD mutations of this gene also exhibit nsl phenotypes, and all of them are more or less impaired in transcriptional activation in vivo. Interestingly, a set of genes affected in the taf1-DeltaTAND mutant is similarly affected in the taf12 HFD mutants but not in the nsl mutants of TBP. Therefore, we discovered that the nsl mutations of these two genes cause lethality in the taf1-DeltaTAND mutant by different mechanisms.

DOI： 10.1093/nar/gkg180

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The orthodenticle-related protein (HpOtx) gene derived from the sea urchin Hemicentrotus pulcherrimus encodes two distinct isoforms, HpOtxE and HpOtxL, which are differentially expressed during early embryogenesis and are driven by TATA-less and TATA-containing promoters, respectively. In order to determine if the TATA element is involved in the establishment of the temporally specific expression profile of the HpOtx gene, reporter genes under the control of modified or wild-type HpOtxE/L promoters were introduced into fertilized eggs. When the activities of the different promoter constructs were examined, we found that deletion of the TATA element from the HpOtxL promoter causes early expression, whereas addition of the TATA element to the HpOtxE promoter causes delayed expression. This suppressive action of the TATA element on transcription from the HpOtxE/L promoters requires the presence of upstream CACGTG elements. These results indicate that the presence or absence of the TATA element determines, at least in part, the expression profile of the HpOtxE/L promoters, in concert with the transcription factor(s) that binds to the upstream CACGTG element. Immunoblot and gel retardation analyses suggest that functional interaction between CACGTG binding factor(s) and TATA factor(s) may be regulated by an unidentified third factor(s) during early embryogenesis in the sea urchin.

DOI： 10.1093/nar/gkf439

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

DOI： 10.1074/jbc.m102416200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The addition of 2,4-dichlorophenoxyacetic acid (2, 4-D) to Vitis sp. cell cultures significantly enhanced the production of quercetin 3,7,4 ' -tri-O-glucoside, 3,7-di-O-glucoside and 3,4-di-O-glucoside from quercetin. This enhancement of glucosylation by 2,4-D was also observed in cell cultures of other plant species. The activity of UDP-glucose: flavonol glucosyltransferase (UFGT) in cell-free extracts of Vitis sp. cell cultures increased approximately 10-fold, 48 h after the addition of 2,4-D to the culture medium. The UFGT activity increased linearly up to 15 h and showed a maximal response to the addition of 10-50 mg/l of 2,4-D at 48 li. The promotive effect of 2,4-D was inhibited by cycloheximide suggesting that de novo protein synthesis was involved in this phenomenon. Interestingly, similar promotive effects on the UFGT activity were observed for other phytohormones such as kinetin and several anti-auxins.

DOI： 10.1263/jbb.91.564

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The general transcription factor TFIID, which is composed of the TATA box-binding protein (TBP) and a set of TBP-associated factors (TAFs), is crucial for both basal and regulated transcription by RNA polymerase II. The N-terminal small segment of yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP function. To understand the physiological role of this inhibitory domain, which is designated as TAND (TAF N-terminal domain), we screened mutations, synthetically lethal with the TAF145 gene lacking TAND (taf145ΔTAND), in Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay. Our screen yielded several recessive nsl (ΔTAND synthetic lethal) mutations, two of which, nsl1-1 and nsl1-2, define the same complementation group. The NSL1 gene was found to be identical to the SPT15 gene encoding TBP. Interestingly, both temperature-sensitive nsl1/spt15 alleles, which harbor the single amino acid substitutions, S118L and P65S, respectively, were defective in transcriptional activation in vivo. Several other previously characterized activation-deficient spt15 alleles also displayed synthetic lethal interactions with taf145ΔTAND, indicating that TAND and TBP carry an overlapping but as yet unidentified function that is specifically required for transcriptional regulation.

DOI： 10.1074/jbc.M008208200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.35% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAG. M-LAO, up to a final concentration of 2.6 muM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alpha IIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4838(00)00229-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

DOI： 10.1074/jbc.m001031200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.120074297

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

<title>ABSTRACT</title>
The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes. We isolated <italic>Saccharomyces cerevisiae</italic> yeast <italic>TAF145</italic> (<italic>yTAF145</italic>) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures. The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reported<italic>yTAF145</italic> mutant (W.-C. Shen and M. R. Green, Cell 90:615–624, 1997). To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of the<italic>TUB2</italic> promoter using a novel mini-<italic>CLN2</italic> hybrid gene reporter system. The results showed that the <italic>yTAF145</italic>mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested. These observations are consistent with the reported yTAF145 dependence of the <italic>CLN2</italic> core promoter in the mutant isolated by Shen and Green, although the <italic>CLN2</italic> core promoter functioned normally in the mutants we report here. These results suggest that different promoters require different yTAF145 functions for efficient transcription. Interestingly, insertion of a canonical TATA element into the TATA-less <italic>TUB2</italic> promoter rescued impaired transcription in the <italic>yTAF145</italic> mutants we studied. It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function.

DOI： 10.1128/mcb.20.7.2385-2399.2000

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The cDNA clones of two isoforms (enzymes I and II) of human placental ecto-ATP diphosphohydrolases have been isolated based on the N-terminal amino acid (aa) sequence of the immunopurified 82 kDa protein and characterized. The cDNA clone encoding enzyme I consists of 2081 nucleotides and the predicted enzyme I consists of 517 aa residues. Enzyme I has a 5'-UTR and an N-terminal II aa sequence that differ from CD39, but the rest of the sequence is the same as CD39, The hydropathy plot indicated that enzyme I has two hydrophobic regions near the N- and C-termini of the molecule. In contrast, enzyme II consists of 1814 nucleotides and the predicted protein consists of 306 aa residues, The sequence of 1-1018 nucleotides of enzyme II is identical to that of enzyme I, but the 1019-1814 nucleotide sequence is different from both enzyme I and CD39, The hydropathy plot indicated that enzyme II has one hydrophobic region near the N-terminus, suggesting that enzyme II is also anchored to the cell membrane. It is, however, likely that some of enzyme II exists as a soluble form in plasma, possibly after proteolytic processing. (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)00751-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Under high shear stress, the binding of von Willebrand factor (VWF) A1-loop domain to platelet glycoprotein (GP) Ib alpha occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1-loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1-loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1-1365 of the subunit). 220 kD Fr (a homodimer of aa residues 1-708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449-728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481-718 of the subunit] and His-rVWF465-728 (a monomer of aa residues 465-728 of the subunit)], and assessed their potency as inhibitors of botrocetin-induced VWF binding to GPIb alpha and high shear stress induced platelet aggregation mediated by intact VWF. All these fragments completely inhibited botrocelin-induced VWF binding to GPIb alpha at a final concentration of 40-200 mu M. The homodimeric A1-loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45-2.0 mu M in the following order: 315 kD Fr greater than or equal to 220 kD Fr much greater than 116 kD Fr. In contrast, the monomeric A1-loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 mu M, and their IC(50)s were 13-39 times higher than those of the homodimers, These results indicate that the homodimeric structure of the A1-loop fragment is important fur optimal molecular interaction with GPIb alpha under high shear stress.

DOI： 10.1046/j.1365-2141.1999.01454.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

TFIID is a multiprotein complex composed of TBP and several TAF(II)s, Small amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAF(II)230 (dTAF(II)230) and yeast TAF(II)145 (yTAF(II)145) bind strongly to TBP and inhibit TBP-DNA interactions. yTAF(II)145 TAND (yTAND) was divided into two subdomains, yTaNDI(10-37) and yTANDII(46-71), that function cooperatively, Here, we identify dTANDII: within the amino terminus of dTAF(II)230 at 118-143 amino acids in addition to dTANDI(18-77), reported previously, dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro. Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP,
Yeast strains containing mutant yTAF(II)145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype, The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37 degrees C, In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAF(II)145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.

DOI： 10.1074/jbc.273.48.32254

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophaa TAF(II)(dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We report here the solution structure of the complex formed by dTAF(II)230 N-terminal region (residues 11-77) and TBP. dTAF(II)230(11-77) comprises three or helices and a beta hairpin, forming a core that occupies the concave DNA-binding surface of TBP. The TBP-binding surface of dTAF(II)230 markedly resembles the minor groove surface of the partially unwound TATA box in the TBP-TATA complex. This protein mimicry of the TATA element surface provides the structural basis of the mechanism by which dTAF(II)230 negatively controls the TATA box-binding activity within the TFIID complex.

DOI： 10.1016/S0092-8674(00)81599-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：F K SCHATTAUER VERLAG GMBH  

The entire cDNA sequences of a never snake venom platelet glycoprotein (GP) Ib-binding protein (BP) composed of an alpha/beta heterodimeric structure, termed mamushigin, from Agkistrodon halys blamhoffii were determined, that include the leader peptides (21/23 amino acid residues) and mature subunits (136/123 amino acid residues). The mature subunits of mamushigin are 37.5% identical, and showed a high degree of similarity (37.7-67.5% identity) with the respective subunits of group W C-type lectins (19). The sequences of the leader peptides of the mamusigin subunits showed the highest similarity (alpha-73.9/beta-82.6%) with those of factor IX/X-BP from Trimeresurus flavoviridis, and the cleavage site residue in both proteins was the same Ala(-1.)
The GPIb-binding specificity of mamushigin is strongly supported by several lines of evidence, but mamushigin can directly aggregate normal platelets, similar to alboaggregin-B (AL-B) (1). This differs from other GPIb-BP's. In mamushigin-treated platelets, serotonin.was not released, and flow cytometric analysis using a monoclonal antibody PAC-1 totally excluded platelet GPIIb/IIIa activation. Mamushigin enhanced platelet aggregation at low-shear stress, and this effect totally disappeared in the presence of GPIb-receptor blockers specific for von Willebrand factor binding, but not by GPIIb/IIIa-receptor blockers. At high-shear stress, mamushigin blocked platelet aggregation in a dose-dependent manner, as seen with other GPIb-BP's. This paper, therefore, describes the cDNA cloning and molecular characterization of mamushigin which has a different effect on platelet aggregation under different shear stress.

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<title>ABSTRACT</title>
The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione<italic>S</italic>-transferase (GST)–Gcn4p fusion protein to components of three different coactivator complexes in <italic>Saccharomyces cerevisiae</italic> cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAF_II20 [yTAF_II20], yTAF_II60, and yTAF_II90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAF_II90, and the yTAF_II20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAF_II proteins are dispensable for activation by Gcn4p in vivo.

DOI： 10.1128/mcb.18.3.1711

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<title>ABSTRACT</title>
The <italic>Drosophila</italic> 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-binding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperature-sensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory domain is involved in TFIIA function.

DOI： 10.1128/mcb.18.2.1003

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DOI： 10.1073/pnas.94.1.85

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The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

DOI： 10.1016/S0092-8674(00)81821-8

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DOI： 10.1038/380316a0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.91.9.3520

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DOI： 10.1038/367484a0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Transcription initiation factor TFIID is a multimeric protein complex that plays a central role in mediating promoter responses to various activators and repressors. To further understand the role of the 85-kDa TFIID subunit (p85), we have cloned the corresponding cDNA with a probe based on an amino acid sequence of the purified protein. The recombinant p85 interacts directly with both the TATA box-binding subunit (TFIID tau or TBP) and the 110-kDa subunit (p110) of TFIID, suggesting that p85 may play a role in helping to anchor p110 within the TFIID complex and, with other studies, that TFIID assembly and function may involve a concerted series of subunit interactions. Interestingly, the carboxy terminus of p85 contains eight of the WD-40 repeats found originally in the beta subunit of G proteins and more recently in other transcriptional regulatory factors. However, truncated p85 lacking all the WD-40 repeats maintained interactions with both TFIID tau and p110. These observations leave open the possibility of a distinct function for the WD-40 repeats, possibly in transducing signals by interactions with transcriptional regulators and/or other components of the basic transcriptional machinery.

DOI： 10.1128/mcb.13.12.7859

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A TATA box-binding initiation factor, TFIID, plays a central role in the transcriptional regulation by activators. Using anti-TFIIDtau (a TATA box-binding component of native TFIID) immunoaffinity chromatography, nine polypeptides (230, 110, 85, 62, 58, 42, 28, 22, and 21 kDa) were identified as native Drosophila TFIID components that are tightly associated with TFIIDtau. To verify the functional activity of the purified TFIID complex, template DNA and other transcription factors were reconstituted with purified TFIID bound to the antibody-Sepharose matrix. Immobilized TFIID mediated not only basal transcription but transcriptional activation by upstream stimulatory factor (USF). On the other hand, recombinant TFIIDtau immobilized on the same antibody-Sepharose matrix could not mediate activation by USF. These results suggest that one or more of these additional polypeptides are required as functional TFIID subunits for activator-dependent transcription in conjunction with TFIIDtau. As further evidence of the relevance of the Drosophila TFIID components identified in this analysis, including the previously unrecognized p230 (Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Cell 66, 563-576), protein blot analysis showed that TFIIDtau interacts specifically and exclusively with p230. This suggests that p230 is an integral subunit of TFIID and that it may play a major role in tethering other subunits to TFIIDtau.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.90.13.5896

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Transcription initiation factor TFIIB recruits RNA polymerase II to the promoter subsequent to interaction with a preformed TFIID-promoter complex. The domains of TFIIB required for binding to the TFIID-promoter complex and for transcription initiation have been determined. The carboxyl-terminal two-thirds of TFIIB, which contains two direct repeats and two basic residue repeats, is sufficient for interaction with the TFIID-promoter complex. An extra 84-residue amino-terminal region, with no obvious known structural motifs, is required for basal transcription activity. Basic residues within the second basic repeat of TFIIB are necessary for stable interaction with the TFIID-promoter complex, whereas the basic character of the first basic repeat is not. Functional roles of other potential structural motifs are discussed in light of the present study.

DOI： 10.1126/science.8332911

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS  

A Drosophila cDNA encoding the largest TFIID subunit (p230) was isolated using a degenerate oligodeoxynucleotide probe based on an amino acid sequence of the purified protein. The entire cDNA sequence contains an open reading frame encoding a polypeptide of 2068 amino acids, corresponding to a calculated molecular mass of 232 kD. The deduced amino acid sequence showed a strong sequence similarity with the protein encoded by a human gene (CCG1) implicated in cell cycle progression through G1, suggesting that p230 may be a target for cell cycle regulatory factors. The recombinant protein expressed in Sf9 cells via a baculovirus vector interacts directly with the TATA box-binding subunit of TFIID (TFIIDtau or TBP) from Drosophila, human, and yeast. Surprisingly, recombinant p230 inhibits the TATA box-binding activity and function of TFIIDtau, suggesting that p230 interactions with TFIIDtau, and possible modulations thereof by other factors may play an important role in TFIID function.

DOI： 10.1101/gad.7.6.1033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.89.24.11809

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.89.7.2839

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

DOI： 10.1271/bbb1961.55.613

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Suspension cultures of a Vitis hybrid converted quercetin to quercetin 3,7,4'-tri-O-glucoside which was characterized by spectroscopy and TLC analysis. Accumulation of quercetin 3,7,4'-triglucoside in the cells was stimulated when cultured in a high concentration of 2,4-dichlorophenoxyacetic acid.

DOI： 10.1016/0031-9422(91)85261-W

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

A cell suspension culture of a Vitis hybrid converted quercetin to six glucosides. Their structures were identified as quercetin 3-O-beta-D-glucopyranoside, 3,4'-di-O-Beta-D-glucopyranoside, quercetin 3,7-di-O-Beta-D-glucopyranoside, isorhamnetin 3-O-Beta-D-glucopyranoside, isorhamnetin 3,4-di-O-beta-D-glucopyranoside, and isorhamnetin 3,7-di-O-beta-D-glucopyranoside by UV, FD-MS, H-1-NMR, C-13-NMR spectroscopy and TLC analysis.
The course of conversion was also investigated and it was shown that quercetin 3-O-glucoside reached the maximum yield of 31% in 24 hr and then gradually disappeared accompanied by the production of quercetin 3,4'-and 3,7-di-O-glucosides. Although the same rise and fall relationship was observed between isorhamnetin 3-O-glucoside and isorhamnetin 3,4'- or 3,7-di-O-glucoside, their conversion ratios were much lower than those of quercetin glucosides.

DOI： 10.1271/bbb1961.54.3283

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Language：Japanese  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN  

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Presentation type：Poster presentation  

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