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PubMed2000

Structure of the central core domain of TFIIEbeta with a novel double-stranded DNA-binding surface.

Human general transcription factor TFIIE consists of two subunits, TFIIEalpha and TFIIEbeta. Recently, TFIIEbeta has been found to bind to the region where the promoter starts to open to be single-stranded upon transcription initiation by RNA polymerase II. Here, the central core domain of human TFIIEbeta (TFIIEbetac) has been identified by a limited proteolysis. This solution structure has been determined by NMR. It consists of three helices with a beta hairpin at the C-terminus, resembling the winged helix proteins. However, TFIIEbetac shows a novel double-stranded DNA-binding activity where the DNA-binding surface locates on the opposite side to the previously reported winged helix motif by forming a positively charged furrow. A model will be proposed that TFIIE stabilizes the preinitiation complex by binding not only to the general transcription factors together with RNA polymerase II but also to the promoter DNA, where double-stranded DNA starts to open to be single-stranded upon activation of the preinitiation complex.

Amino Acid Sequence↗
PubMed2002

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts.

Core binding factor alpha 1 (Cbfa1) is an osteoblast-specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic-differentiating agents is still unclear. In this study, we assessed the effects of 1,25-(OH)(2)-D3 (D3), ascorbic acid, bone morphogenetic protein-2 (BMP-2), dexamethasone (Dex), and transforming growth factor-beta (TGF-beta) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT-PCR) in an in vitro model of osteoblast differentiation. TGF-beta increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6-fold in a time-dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time-dependent fashion by up to 4.6-fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP-2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2-fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5-fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14-fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation.

Adult↗
PubMed2005

Beta-catenin and BMP-2 synergize to promote osteoblast differentiation and new bone formation.

Mutations of critical components of the Wnt pathway profoundly affect skeletal development and maintenance, probably via modulation of beta-catenin signaling. We tested the hypothesis that beta-catenin is involved in mesenchymal lineage allocation to osteogenic cells using a beta-catenin mutant with constitutive transcriptional activity (DeltaN151). Although this stable beta-catenin had no effects by itself on osteogenic differentiation of multipotent embryonic cell lines, it synergized with bone morphogenetic protein-2 (BMP-2) resulting in dramatic stimulation of alkaline phosphatase activity, osteocalcin gene expression, and matrix mineralization. Likewise, DeltaN151 and BMP-2 synergistically stimulated new bone formation after subperiosteal injection in mouse calvaria in vivo. Conversely, DeltaN151 prevented adipogenic differentiation from pre-adipocytic or uncommitted mesenchymal cells in vitro. Intriguingly, the synergism with BMP-2 on gene transcription occurred without altering expression of Cbfa1/Runx2, suggesting actions independent or downstream of this osteoblast-specific transcription factor. Thus, beta-catenin directs osteogenic lineage allocation by enhancing mesenchymal cell responsiveness to osteogenic factors, such as BMP-2, in part via Tcf/Lef dependent mechanisms. In vivo, this synergism leads to increased new bone formation.

Adipocytes↗
PubMed2000

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding.

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.

Amino Acid Motifs↗
PubMed2000

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

Animals↗
PubMed2005

Bone morphogenetic protein-2 suppresses collagenase-3 promoter activity in osteoblasts through a runt domain factor 2 binding site.

Transforming growth factor-beta (TGFbeta) superfamily of growth factors, which include bone morphogenetic proteins (BMPs), have multiple effects in osteoblasts. In this study, we examined the regulation of collagenase-3 promoter activity by BMP-2 in osteoblast-enriched (Ob) cells from fetal rat calvariae. BMP-2 suppressed the activity of a -2 kb collagenase-3 promoter/luciferase recombinant in a time- and dose-dependent manner. The BMP-2 effect on the collagenase-3 promoter was further tested in several collagenase-3 promoter deletion constructs and it was narrowed down to a -148 to -94 nucleotide segment of the promoter containing a runt domain factor 2 (Runx2) site at nucleotide -132 to -126. The effect of BMP-2 was obliterated in a collagenase-3 promoter/luciferase construct containing a mutated Runx2 (mRunx2) sequence indicating that the Runx2 site mediates the BMP-2 response. Electrophoretic mobility shift assays, using nuclear extracts from control and BMP-2-treated Ob cells, indicated that the Runx2 protein is a component of the specific DNA-protein complex formed on the Runx2 site and that the BMP-2 effect may be associated with minor protein modifications rather than major changes in the composition of specific proteins interacting with the Runx2 site. We confirmed that other members of the TGFbeta family can down-regulate the collagenase-3 promoter by showing that TGFbeta1 also suppresses the promoter activity in a time- and dose-dependent manner. In conclusion, this study demonstrates that BMP-2 and TGFbeta1 suppress collagenase-3 promoter activity in osteoblasts and establishes a link between BMP-2 action and collagenase-3 expression via Runx2, a major regulator of osteoblast formation and function.

Animals↗
PubMed2006

The RUNX3 tumor suppressor upregulates Bim in gastric epithelial cells undergoing transforming growth factor beta-induced apoptosis.

Genes involved in the transforming growth factor beta (TGF-beta) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUNX3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3-/- gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-beta. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids 1 to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3-/- mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim-/- gastric epithelium. We confirmed comparable expression of TGF-beta1 and TGF-beta receptors between wild-type and Runx3-/- gastric epithelia and reduction of Bim in TGF-beta1-/- stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-beta-induced apoptosis.

Active Transport, Cell Nucleus↗
PubMed2001

Characterization of the upstream mouse Cbfa1/Runx2 promoter.

Cbfa1 (or Runx2/AML-3/PEPB2alpha) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 5'-flanking region of the Cbfa1 gene containing its "bone-related" or P1 promoter and exon 1. We identified additional coding sequence in exon 1 and splice donor sites that potentially give rise to a novel Cbfa1 isoform containing an 18 amino acid insert. In addition, primer extension mapping identified in the Cbfa1 promoter a minor mRNA start site located approximately 0.8 kb 5' upstream of the ATG encoding the MASN/p57 isoform and approximately 0.4 kb upstream of the previously reported start site. A luciferase reporter construct containing 1.4 kb of the mouse Cbfa1 promoter was analyzed in Ros 17/2.8 and MC3T3-E1 osteoblast cell lines that express high levels of Cbfa1 transcripts. The activity of this construct was also examined in non-osteoblastic Cos-7 and NIH3T3 cells that do not express Cbfa1 and mesenchymal-derived cell lines, including CH3T101/2, C2C12, and L929 cells, that express low levels of mature Cbfa1 transcripts. The 1.4 kb 5' flanking sequence of the Cbfa1 gene directed high levels of transcriptional activity in Ros 17/2.8 and MC3T3-E1 osteoblasts compared to non-osteoblasts Cos-7 cells, but this construct also exhibited high levels of expression in C310T1/2, L929, and C2C12 cells as well as NIH3T3 cells. In addition, Cbfa1 mRNA expression, but not the activity of the Cbfa1 promoter, was upregulated in a dose-dependent manner in pluripotent mesenchymal C2C12 by bone morphogenetic protein-2 (BMP-2). These data indicate that Cbfa1 is expressed in osteogenic as well as non-osteogenic cells and that the regulation of Cbfa1 expression is complex, possibly involving both transcriptional and post-transcriptional mechanisms. Additional studies are needed to further characterize important regulatory elements and to identify additional regions of the promoter and/or post-transcriptional events responsible for the cell-type restricted regulation of Cbfa1 expression.

3T3 Cells↗
PubMed2006

C-type natriuretic peptide enhances osteogenic protein-1-induced osteoblastic cell differentiation via Smad5 phosphorylation.

In the present study, we examined the hypothesis that the C-type natriuretic peptide (CNP) enhances osteogenic protein-1 (OP-1) action in stimulating osteoblastic cell differentiation in primary cultures of fetal rat calvaria cell (FRC). CNP enhanced synergistically the OP-1-induced Alkaline Phosphatase (AP) activity and mineralized bone nodule formation in a dose- and time-dependent manner. To examine possible mechanism of the synergy between OP-1 and CNP, the expression levels of key BMP receptors and signaling molecules were examined. Western blot analysis showed that BMPR-IB and -II receptor protein expression was not affected by CNP alone, but was stimulated by OP-1 alone. The combination of OP-1 and CNP did not further increase their protein levels. The Runx2 protein expression level was not altered by CNP alone, but was elevated by OP-1 alone, and was slightly reduced by the combination. The Smad5 protein expression level was slightly decreased by CNP alone, but was stimulated by OP-1 alone, and was not further stimulated by the combination. Smad5 phosphorylation was not stimulated by CNP alone, but was stimulated significantly by OP-1 alone. The combination of OP-1 and CNP further stimulated the OP-1-induced Smad5 phosphorylation. Thus, one mechanism of the observed synergy between OP-1 and CNP involves enhancement of the Smad5 phosphorylation.

Alkaline Phosphatase↗
PubMed2007

Bone morphogenetic protein 2 activates Smad6 gene transcription through bone-specific transcription factor Runx2.

BMP-2 plays an essential role in osteoblast and chondrocyte differentiation, but its signaling mechanism has not been fully defined. In the present studies, we investigated the mechanism through which BMP-2 activates the Smad6 gene. A -2006/+45 Smad6 promoter-luciferase construct was generated along with deletions and Runx2 binding site mutations to examine the role of Smad1 and Runx2 signaling following BMP-2 stimulation in osteoblasts. Transfection of Runx2 or treatment with BMP-2-stimulated promoter activity of the -2006/+45 and -1191/+45 reporters but not the -829/+45 and -374/+45 reporters. No Smad1/5 binding site is present in the -1191/-829 region of the Smad6 promoter. Mutation of the OSE2-a site (-1036/-1031) completely abolished the stimulatory effect of Runx2 as well as BMP-2 on the -2006/+45 and -1191/+45 Smad6 reporters. Gel shift and chromatin immunoprecipitation (ChIP) assays showed that Runx2 binds the OSE2-a element. ChIP assays demonstrated that Smad1 also interacts with the OSE2-a site at the Smad6 promoter through Runx2. The protein degradation of Runx2 is mediated by the E3 ubiquitin ligase Smurf1. In the present studies, we found that Smurf1 binds the OSE2-a site through Runx2 and inhibits Smad6 gene transcription. Treatment with BMP-2 and transfection of Smad1 abolished Smurf1 binding to the OSE2 site. These results show that Smad1 binding excludes Smurf1 interaction with the OSE2 site and promotes Smad6 gene transcription.

Animals↗
PubMed2004

Recombinant human bone morphogenetic protein-2 promotes Indian hedgehog-mediated osteo-chondrogenic differentiation of a human chondrocytic cell line in vivo and in vitro.

We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.

Animals↗
PubMed2001

Differential regulation of the two principal Runx2/Cbfa1 n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype.

Cbfa1/Runx2 is a transcription factor essential for bone formation and osteoblast differentiation. Two major N-terminal isoforms of Cbfa1, designated type I/p56 (PEBP2aA1, starting with the sequence MRIPV) and type II/p57 (til-1, starting with the sequence MASNS), each regulated by distinct promoters, are known. Here, we show that the type I transcript is constitutively expressed in nonosseous mesenchymal tissues and in osteoblast progenitor cells. Cbfa1 type I isoform expression does not change with the differentiation status of the cells. In contrast, the type II transcript is increased during differentiation of primary osteoblasts and is induced in osteoprogenitors and in premyoblast C2C12 cells in response to bone morphogenetic protein-2. The functional equivalence of the two isoforms in activation and repression of bone-specific genes indicates overlapping functional roles. The presence of the ubiquitous type I isoform in nonosseous cells and before bone morphogenetic protein-2 induced expression of the type II isoform suggests a regulatory role for Cbfa1 type I in early stages of mesenchymal cell development, whereas type II is necessary for osteogenesis and maintenance of the osteoblast phenotype. Our data indicate that Cbfa1 function is regulated by transcription, cellular protein levels, and DNA binding activity during osteoblast differentiation. Taken together, our studies suggest that developmental timing and cell type- specific expression of type I and type II Cbfa isoforms, and not necessarily molecular properties or sequences that reside in the N-terminus of Cbfa1, are the principal determinants of the osteogenic activity of Cbfa1.

Animals↗
PubMed2005

Identification of functional elements in the murine Gabp alpha/ATP synthase coupling factor 6 bi-directional promoter.

The GA-repeat binding protein (GABP) is a ubiquitous transcription factor involved in transcriptional regulation of genes encoding proteins involved in a variety of cellular processes including adipocyte differentiation, mitochondrial respiration, and neuromuscular signaling. GABP is composed of two subunits; the GABP alpha subunit is a member of the Ets-family of transcription factors, and the unrelated ankyrin repeat containing GABP beta subunit. We previously identified a bidirectional promoter directing the expression of Gabpa (GAA) gene in one direction and ATP Synthase Coupling Factor 6 (Atp5j) (CF6) gene in the other [Chinenov, Y., Coombs, C. and Martin, M. E., 2000a. "Isolation of a bi-directional promoter directing the expression of the mouse GABP alpha and ATP Synthase Coupling Factor 6 genes. Gene 261:311-320.]. In this study we characterize sequence elements and regulatory factors contributing to the promoter activities of the GAA/CF6 bidirectional promoter. The core of the GAA/CF6 bidirectional promoter is retained within a 400 bp sequence and contains four GABP binding sites, a Sp1/3 binding site and an YY1 binding site. Site-directed mutagenesis demonstrated that while no single factor binding site was essential for promoter activity in either direction, the GA1 site located proximal to the previously mapped transcription start sites functioned cooperatively with the other GABP binding sites and with the Sp1/3 and YY1 sites to provide transcriptional activation of the GAA and CF6 promoters. The other GABP sites and the Sp1/3 and YY1 binding sites were functionally redundant for basal promoter activities in both directions. Electrophoretic mobility shift assays identified multiple DNA-protein complexes containing GABP alpha, GABP beta, Sp1, Sp3 or YY1 proteins, including one ternary complex containing GABP alpha, GABP beta and Sp1 proteins. Binding of GABP to the GAA/CF6 bi-directional promoter provides the potential for autoregulation of GABP alpha expression and confirms the importance of GABP in the coordinate expression of respiratory chain components.

Animals↗
PubMed2002

Regulatory mechanisms of osteoblast and osteoclast differentiation.

Bone is continuously destroyed and reformed to maintain constant bone volume and calcium homeostasis in vertebrates throughout their lives. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Recent developments in bone cell biology have greatly changed our conceptions of the regulatory mechanisms of the differentiation of osteoblasts and osteoclasts. Bone morphogenetic proteins (BMPs) play critical roles in osteoblast differentiation. The discovery of Smad-mediated signals revealed the precise functions of BMPs in osteoblast differentiation. Transcription factors, Runx2 and Osterix, are found to be essential molecules for inducing osteoblast differentiation, as indicated by the fact that both Runx2-null mice and Osterix-null mice have neither bone tissue nor osteoblasts. Smad transcriptional factors are shown to interact with other transcription regulators, including Runx2. Also, the recent discovery of receptor activator of NF-kappaB ligand (RANKL)-RANK interaction confirms the well-known hypothesis that osteoblasts play an essential role in osteoclast differentiation. Osteoblasts express RANKL as a membrane-associated factor. Osteoclast precursors that express RANK, a receptor for RANKL, recognize RANKL through the cell-cell interaction and differentiate into osteoclasts. Recent studies have shown that lipopolysaccharide and inflammatory cytokines such as tumor necrosis factor receptor-alpha and interleukin I directly regulate osteoclast differentiation and function through a mechanism independent of the RANKL-RANK interaction. Transforming growth factor-beta super family members and interferon-gamma are also shown to be important regulators in osteoclastogenesis. These findings have opened new areas for exploring the molecular mechanisms of osteoblast and osteoclast differentiation.

Animals↗
PubMed2004

Runx2 deficiency in chondrocytes causes adipogenic changes in vitro.

Runx2 (runt-related transcription factor 2) is an important transcription factor for chondrocyte differentiation as well as for osteoblast differentiation. To investigate the function of Runx2 in chondrocytes, we isolated chondrocytes from the rib cartilage of Runx2-deficient (Runx2-/-) mice and examined the effect of Runx2 deficiency on chondrocyte function and behavior in culture for up to 12 days. At the beginning of the culture, Runx2-/- chondrocytes actively proliferated, had a polygonal shape and expressed type II collagen; these are all characteristics of chondrocytes. However, they gradually accumulated lipid droplets that stained with oil red O and resembled adipocytes. Northern blot analysis revealed that the expression of adipocyte-related differentiation marker genes including PPAR gamma (peroxisome proliferator-activated receptor gamma), aP2 and Glut4 increased over time in culture, whereas expression of type II collagen decreased. Furthermore, the expression of Pref-1, an important inhibitory gene of adipogenesis, was remarkably decreased. Adenoviral introduction of Runx2 or treatment with transforming growth factor-beta, retinoic acid, interleukin-1 beta, basic fibroblast growth factor, platelet-derived growth factor or parathyroid hormone inhibited the adipogenic changes in Runx2-/- chondrocytes. Runx2 and transforming growth factor-beta synergistically upregulated interleukin-11 expression, and the addition of interleukin-11 to the culture medium reduced adipogenesis in Runx2-/- chondrocytes. These findings indicate that depletion of Runx2 resulted in the loss of the differentiated phenotype in chondrocytes and induced adipogenic differentiation in vitro, and show that Runx2 plays important roles in maintaining the chondrocyte phenotype and in inhibiting adipogenesis. Our findings suggest that these Runx2-dependent functions are mediated, at least in part, by interleukin-11.

Adaptor Protein Complex 2↗
PubMed1998

High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.

BACKGROUND: Transcriptional initiation and elongation provide control points in gene expression. Eukaryotic RNA polymerase II subunit 9 (RPB9) regulates start-site selection and elongational arrest. RPB9 contains Cys4 Zn(2+)-binding motifs which are conserved in archaea and homologous to those of the general transcription factors TFIIB and TFIIS. RESULTS: The structure of an RPB9 domain from the hyperthermophilic archaeon Thermococcus celer was determined at high resolution by NMR spectroscopy. The structure consists of an apical tetrahedral Zn(2+)-binding site, central beta sheet and disordered loop. Although the structure lacks a globular hydrophobic core, the two surfaces of the beta sheet each contain well ordered aromatic rings engaged in serial edge-to-face interactions. Basic sidechains are clustered near the Zn(2+)-binding site. The disordered loop contains sidechains conserved in TFIIS, including acidic residues essential for the stimulation of transcriptional elongation. CONCLUSIONS: The planar architecture of the RPB9 zinc ribbon-distinct from that of a conventional globular domain-can accommodate significant differences in the alignment of polar, non-polar and charged sidechains. Such divergence is associated with local and non-local changes in structure. The RPB9 structure is distinguished by a fourth beta strand (extending the central beta sheet) in a well ordered N-terminal segment and also differs from TFIIS (but not TFIIB) in the orientation of its apical Zn(2+)-binding site. Cys4 Zn(2+)-binding sites with distinct patterns of polar, non-polar and charged residues are conserved among unrelated RNAP subunits and predicted to form variant zinc ribbons.

Amino Acid Sequence↗
PubMed1981

Interaction of the sigma factor and the nusA gene protein of E. coli with RNA polymerase in the initiation-termination cycle of transcription.

The nusA gene protein of E. coli is involved in regulating termination of transcription in vivo. In vitro it causes termination of transcription in the tR2 region of the PR operon of bacteriophage lambda. We have now used a nusA-Sepharose affinity column and gradient sedimentation experiments to show that the nusA protein binds directly, with great specificity, and with equimolar stoichiometry to the E. coli RNA polymerase core enzyme beta beta' alpha 2. The RNA polymerase sigma subunit is able to displace the nusA protein from a beta beta' alpha 2-nusA complex, regenerating RNA polymerase holoenzyme beta beta alpha 2-sigma able selectively to initiate transcription at promoter sites. It is proposed that beta beta' alpha 2-nusA and beta beta' alpha 2-sigma are complementary forms of RNA polymerase that interchange with one another in the initiation-termination cycle of transcription.

Bacterial Proteins↗
PubMed2006

Runx3 expression during mouse tongue and palate development.

Studies of molecular mechanisms underlying the development of the mammalian oral mucosa have revealed a major involvement of transforming growth factor beta (TGF-beta) and bone morphologic protein (BMP) signaling pathways. Here, we examined the expression of a downstream target of TGF-beta and BMPs, Runx3, in oral mucosa. Runx3 is a runt-related transcription factor that acts as a gastric tumor suppressor and regulator of growth and differentiation in mammalian gastric epithelial cells. Another member of the Runx family in C. elegans, run, is involved in the development of a functional hypodermis and gut. In this report, we examined Runx3 expression using reverse transcription-polymerase chain reaction, immnunohistochemistry, and in situ hybridization and found that Runx3 is expressed in the tongue and palate epithelium of mouse embryos from embryonic day 12.5 to 16.5. The functional relationship between Runx3 and TGF-beta/BMPs signaling in tongue and palate development is discussed.

Animals↗