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At least 73 records · Page 4Linked to original sources

PubMed2003

The small nuclear RNA-activating protein 190 Myb DNA binding domain stimulates TATA box-binding protein-TATA box recognition.

Human U6 small nuclear RNA (snRNA) gene transcription by RNA polymerase III requires cooperative promoter binding involving the snRNA-activating protein complex (SNAP(c)) and the TATA-box binding protein (TBP). To investigate the role of SNAP(c) for TBP function at U6 promoters, TBP recruitment assays were performed using full-length TBP and a mini-SNAP(c) containing SNAP43, SNAP50, and a truncated SNAP190. Mini-SNAP(c) efficiently recruits TBP to the U6 TATA box, and two SNAP(c) subunits, SNAP43 and SNAP190, directly interact with the TBP DNA binding domain. Truncated SNAP190 containing only the Myb DNA binding domain is sufficient for TBP recruitment to the TATA box. Therefore, the SNAP190 Myb domain functions both to specifically recognize the proximal sequence element present in the core promoters of human snRNA genes and to stimulate TBP recognition of the neighboring TATA box present in human U6 snRNA promoters. The SNAP190 Myb domain also stimulates complex assembly with TBP and Brf2, a subunit of a snRNA-specific TFIIIB complex. Thus, interactions between the DNA binding domains of SNAP190 and TBP at juxtaposed promoter elements define the assembly of a RNA polymerase III-specific preinitiation complex.

DNA-Binding Proteins↗

PubMed2006

JAK1 N-terminus binds to conserved Box 1 and Box 2 motifs of cytokine receptor common beta subunit but signal activation requires JAK1 C-terminus.

The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.

Amino Acid Motifs↗

PubMed2006

Bacterial count comparisons on examination gloves from freshly opened boxes versus nearly empty boxes and from examination gloves before treatment versus after dental dam isolation.

Use of gloves in dentistry is primarily for protection of the healthcare worker, yet little information is available regarding potential patient issues such as microbial contamination of gloves before and during use. The purposes of this study were to compare gloves from newly opened boxes with those from boxes that had been in dental operatories until they were nearly empty and to determine if gloves are contaminated though diagnostic procedures and rubber dam placement. Eight endodontic residents provided samples by streaking gloved fingers on nutrient agar plates. Results showed no statistically significant difference between counts from new boxes versus nearly empty boxes (n = 32, p < 0.6216). Gloves after rubber dam placement yielded a mean colony count of 158 versus a 1.5 mean of fresh gloves (n = 64, p < 0.0001). These results suggest that the use of new gloves before opening a tooth for endodontic therapy may be warranted.

Dental Offices↗

PubMed1992

Combination of H-box [CCTACC(N)7CT] and G-box (CACGTG) cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid.

The phenylpropanoid pathway intermediate p-coumaric acid (4-CA) stimulates expression of the bean (Phaseolus vulgaris L.) chalcone synthase (malonyl-CoA:4-coumaroyl-CoA, EC 2.3.1.74) chs15 gene promoter in electroporated protoplasts of alfalfa (Medicago sativa L.). We have analyzed the effects of 5' deletions, mutations, and competition with promoter sequences in trans on the expression of a chs15 promoter-chloramphenicol acetyltransferase gene fusion in elicited alfalfa protoplasts. Two distinct sequence elements, the H-box (consensus CCTACC(N)7CT) and the G-box (CACGTG), are required for stimulation of the chs15 promoter by 4-CA. Furthermore, a 38-base-pair chs15 promoter sequence containing both cis elements conferred responsiveness to 4-CA on the cauliflower mosaic virus 35S minimal promoter. The H-box and G-box in combination establish the complex developmental pattern of chs15 expression and are also involved in stress induction. Hence, potential internal pathway regulation through feed-forward stimulation by 4-CA operates by modulation of the signal pathways for developmental and environmental regulation.

Acyltransferases↗

PubMed2003

Drosophila securin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with Cyclin A degradation.

Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM. Consistent with this proposal and with results from yeast and vertebrates, we show here that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, we also show that PIM contains a KEN-box, which is required for mitotic degradation in addition to the D-box, and that sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals.

Amino Acid Motifs↗

PubMed1996

Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism.

Amino Acid Sequence↗

PubMed1994

Interaction between the N-terminal domain of the 230-kDa subunit and the TATA box-binding subunit of TFIID negatively regulates TATA-box binding.

Transcription initiation factor TFIID plays a central role in transcriptional regulation. Drosophila TFIID is a multimeric protein consisting of the TATA box-binding polypeptide (TBP) and a number of tightly associated polypeptides. Previously, the largest subunit of TFIID (p230) was cloned and demonstrated to inhibit the TATA-box binding of TBP in the absence of other subunits. Here we demonstrate that p230 contains at least two sites of interaction with TBP and that the N-terminal site mediates both strong physical interactions with TBP and inhibition of the TBP function. A detailed mutagenesis study shows that the inhibitory domain is indistinguishable from the strong TBP-binding domain, thus indicating that interaction of the p230 N-terminal region with TBP may directly control TATA-box binding.

Amino Acid Sequence↗

PubMed1998

Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase.

BACKGROUND: In the ubiquitin-dependent proteolysis pathway, a ubiquitin ligase (E3) is responsible for substrate selectivity and timing of degradation. A novel E3, SCF (Skp1-Cullin-1/Cdc53-F-box) plays a pivotal role in cell cycle progression. In fission yeast, F-box/WD-repeat protein Pop1 regulates the level of the CDK (cyclin-dependent kinase) inhibitor Rum1 and the S phase regulator Cdc18. RESULTS: We have cloned and characterized the pop2+ gene which encodes the Pop1-related F-box/WD-repeat protein. Pop2 plays a role which overlaps with Pop1 in the degradation of Rum1 and Cdc18. However, these two proteins are not functional homologues. Pop1 and Pop2 form hetero-as well as homo-dimers in the cell. We have analysed two fission yeast cullin members and found that cullin-1 functions as a component of SCFPop1,2, whilst cullin-3 is involved in the distinct stress-response pathway. CONCLUSIONS: Fission yeast SCF is composed of Pop1 and Pop2, two structurally related but functionally independent F-box/WD-repeat proteins. By forming three distinct complexes, SCFPop1/Pop1, SCFPop1/Pop2 and SCFPop2/Pop2, SCF has evolved a sophisticated mechanism to control the level of Rum1 and Cdc18. Fission yeast SCF also contains cullin-1 as a universal scaffold and each cullin member plays a distinct biological role.

Amino Acid Sequence↗

PubMed1995

Reporter gene expression upon stable transfection when only a TATA box or a TATA box plus Sp1 sites are present 5' to the gene.

Episomal plasmids for stable transfection of mammalian cell cultures were constructed that have a G418-resistance (neo) gene immediately downstream of a highly truncated promoter. These plasmids had a function hygromycin-resistance gene (hyg) as a selectable marker. Surprisingly, in LTK- cells, but not HeLa cells, stably transfected with these BK virus-based plasmids having no promoter elements adjacent to the neo gene, readthrough transcription, probably from about 1 kb upstream, gave almost as efficient expression of the neo gene as of the hyg gene with a full-length promoter immediately upstream. When the transfecting plasmids contained Epstein-Barr virus (EBV) DNA sequences for episomal maintenance and had multiple Sp1 sites and a TATA box as the only promoter elements 5' to the neo gene, only about 3-9% of HeLa transfectants were G418 resistant (G418R). In transfections with analogous plasmids lacking these promoter elements 5' to the neo gene, no G418R colonies were seen. The establishment of the G418R phenotype probably required integration of plasmid DNA into favorable chromosomal sites and was aided by the presence of the TATA box plus Sp1 sites as a subminimal promoter. The absence of detectable G418-resistance in most of the HeLa transfectant clones obtained with EBV-type plasmids, even at a high plasmid copy number and even when a TATA box and six Sp1 sites were present immediately upstream of the neo gene, indicates that these elements do not suffice for appreciable gene expression in vivo and that this is a suitable model system for studying DNA rearrangements that can potentiate expression of the neo gene.

Animals↗

PubMed1994

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

Adenovirus E1A Proteins↗

PubMed1998

RNA-binding proteins in mouse oocytes and embryos: expression of genes encoding Y box, DEAD box RNA helicase, and polyA binding proteins.

Growth and differentiation of early embryos depends almost entirely on information which is maternally inherited in the form of macromolecules accumulated by the female gamete during its growth phase. Most of the maternal mRNAs synthesized by growing oocytes are not immediately recruited onto polysomes but are stored as translationally dormant messenger ribonucleoprotein (mRNP) particles. mRNA binding proteins which have been associated with masked mRNP complexes in Xenopus oocytes fall into two main categories, those having affinity for a variety of RNA sequences (members of the Y box and DEAD box RNA helicase families) and those which interact more specifically with 3' polyA tails (the polyA binding proteins or PABPs). The objective of this study was to determine whether mouse oocytes and embryos express sequences encoding a Y box protein, (MSY1); on RNA helicase, (RCK/p54); and a universally expressed PABP and testis specific isoform (PABP1 and PABPt, respectively). RNAs were amplified by RT/PCR and the identities of targeted cDNAs were confirmed by restriction analysis and/or direct sequencing. Relative steady state levels and time courses of accumulation/decay were compared by Northern hybridization. All of the sequences are transcribed as maternal mRNAs. MSY1 transcripts accumulated during the growth phase appear to be degraded in parallel with the bulk of maternal mRNAs by the mid-late two-cell stage. RCK/p54 mRNAs are most abundant in growing oocytes; steady state levels decline in primary and secondary oocytes, and degradation appears to be complete by the mid-late two-cell stage. Zygotic transcription of MSY1 and RCK/p54 is evident in four-cell stage embryos. Most of the PABP1 message accumulated by growing oocytes decays during meiotic maturation with transcription resuming in two-cell embryos. PABPt is expressed at very low levels in oocytes and embryos. Based on the temporal patterns of expression and the reported activities of homologous sequences in other systems, we suggest that these RNA binding proteins may participate in the post-transcriptional regulation of gene expression during the period of maternal control of development in the mouse.

Amino Acid Sequence↗

PubMed1985

Homeo box gene complex on mouse chromosome 11: molecular cloning, expression in embryogenesis, and homology to a human homeo box locus.

The homeo box is a 180 bp protein-coding domain found within homeotic genes of Drosophila and conserved in a variety of invertebrate and vertebrate species. It has been suggested that the mammalian homeo box sequences may play a role in controlling pattern formation during embryogenesis. We report findings that support this hypothesis. We have cloned three overlapping recombinant phage clones that cover a region of mouse chromosome 11 that contains a cluster of four homeo boxes (the Hox-2 locus). This locus encodes multiple transcripts that are expressed during embryogenesis. Forty kilobases of the Hox-2 region is devoid of repetitive elements and shows extensive homology with the human Hox-2 locus. These results provide direct evidence for genetic expression during embryonic development, a conserved organization in comparison to the cognate human locus, and a complexity of organization and transcript expression similar to that found in Drosophila.

Animals↗

PubMed2004

Type I MADS-box genes have experienced faster birth-and-death evolution than type II MADS-box genes in angiosperms.

Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADS-box genes from the Arabidopsis and rice genomes. Plant MADS-box genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in Arabidopsis. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and approximately 15-20 type II genes in the most recent common ancestor of Arabidopsis and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes.

Arabidopsis↗

PubMed2000

Erythroid Kruppel-like factor is recruited to the CACCC box in the beta-globin promoter but not to the CACCC box in the gamma-globin promoter: the role of the neighboring promoter elements.

The programmed expression of the five beta-like globin genes (epsilon, (A)gamma, (G)gamma, delta, and beta) is characterized by a series of switches that are developmentally regulated. The (A)gamma- and (G)gamma- (fetus) to beta-globin (adult) switch depends on transcription factor erythroid Krüppel-like factor (EKLF), which, like Sp1, binds to CACCC boxes. EKLF is essential for the expression of the beta-globin but not the gamma-globin gene. Because both gamma-globin and beta-globin promoters contain the CACCC box, and their promoter elements are similar, it is not known why the two promoters behave so differently. In this report, we searched for the functional differences between the two promoters by studying their ability to recruit EKLF. We used the in vivo PIN*POINT assay to show that EKLF is recruited to the beta-globin promoter but not to the gamma-globin promoter. We show that this selectivity is a result of differences in surrounding promoter elements and not CACCC box alone. One of the differences between the two promoters with a functional consequence is the CCTTG repeat that is present in the gamma-globin promoter but not in the beta-globin promoter. The repeat, when inserted in the beta-globin promoter, decreases EKLF recruitment to and activity of the beta-globin promoter, suggesting that the repeat functions as a suppressor element. The CCTTG repeat can also suppress the SV40 promoter in cis, and the suppressor factor binding to the repeat can be squelched with a plasmid containing a high copy number of the repeat. These findings may have implications in designing drug targets for treatment of beta-globin disorders.

Animals↗

PubMed1994

Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells.

The molecular mechanisms by which expression of a gene is down-regulated after differentiation of F9 embryonal carcinoma cells into parietal endoderm-like cells was studied by characterizing the cis- and trans-regulatory elements of the gb110 gene. This gene encodes a putative RNA helicase, and its expression is down-regulated when F9 cells are differentiated with retinoic acid and cyclic AMP. The 5'-flanking region of the gene has all of the features of a GC-rich island promoter and seems to play only a minor role, if any, in the regulated expression. A 133-bp enhancer in the first intron was identified by transient chloramphenicol acetyltransferase assays that activated expression in undifferentiated F9 cells about 50- to 100-fold. As this enhancer was not active in differentiated F9 cells, it seems to be the prime mediator of the differentiation-specific down-regulation of the gb110 gene. Four different protein-binding sites, three of which contain GC- and GT-box motifs, were identified in the enhancer element. The fourth site, interacting with previously described transcription factor FTZ-F1/ELP, seems to be of minor importance for the activity of the enhancer. Mutational analysis showed that the cooperative interaction of several most likely related proteins with the three GC- and GT-box motifs was required for full enhancer activity. On the basis of their binding properties, at least two of these proteins seem to be identical or closely related to ubiquitous transcription factor Sp1. One of the GT-box-binding proteins was present in undifferentiated F9 cells but not, however, in its differentiated derivatives. The cell specificity of this transcription factor explains why the gb110 gene is not expressed or expressed only at low levels in parietal endoderm-like cells.

Animals↗

PubMed2001

Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing.

Mutations in the 5' portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (A0) in the 3' region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5' and 3' ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box A' region is necessary for site 1 cleavage, and the 3' end of box A' and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5' and 3' ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage.

Animals↗

PubMed1998

Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors.

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.

DNA-Binding Proteins↗

PubMed1997

Drosophila TAF(II)230 and the transcriptional activator VP16 bind competitively to the TATA box-binding domain of the TATA box-binding protein.

The transcription initiation factor TFIID, consisting of the TATA box-binding protein (TBP) and many TBP-associated factors (TAFs), plays a central role in both basal and activated transcription. An intriguing finding is that the 80-residue N-terminal region of Drosophila TAF(II)230 [dTAF(II)230-(2-81)] can bind directly to TBP and inhibit its function. Here, studies with mutated forms of TBP demonstrate that dTAF(II)230-(2-81) binds to the concave surface of TBP, which is important for TATA box binding. Previously, it was reported that a point mutation (L114K) on this concave surface destroys the ability of TBP to bind VP16 and to mediate VP16-dependent activation in vitro, but has no effect on basal transcription. Importantly, the same TBP mutation eliminates TBP binding to dTAF(II)230-(2-81). Consistent with these effects of the L114K mutation, dTAF(II)230-(2-81) and the VP16 activation domain compete for binding to wild-type TBP. These results indicate that transcriptional regulation may involve, in part, competitive interactions between transcriptional activators and TAFs on the TBP surface.

Amino Acid Sequence↗