Purification of human transcription factor IIIC and its binding to the gene for ribosomal 5S RNA

Schneider, Hans-Jochen; Waldschmidt, Rainer; Jahn, Dieter; Seifart, Klaus H.

doi:10.1093/nar/17.13.5003

Cited by 64 publications

(34 citation statements)

References 39 publications

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“…27,28 Endogenous DEDD has also been found to be mainly localized to the cytoplasm and is translocated to the nucleus upon stimulation of CD95/Fas. 16 Our finding that co-expression of hTFIIIC102 with either DEDD or FLAME-3 appears to induce translocation of hTFIIIC102 to the nucleus, suggests that the DEDD proteins may regulate hTFIIIC102 transport across the nuclear membrane under normal physiological circumstances.…”

Section: Discussionmentioning

confidence: 99%

Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC

et al. 2002

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Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-kB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DEDcontaining protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast twohybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD-and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-kB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.

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Section: Discussionmentioning

confidence: 99%

Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC

et al. 2002

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“…Later, human TFIIIC-activity was chromatographically separated into two fractions that either comprised the DNAbinding activity (TFIIIC2) or that contained an essential, yet not primarily DNA-binding activity (TFIIIC1). 1,42 The six subunits constituting the DNA-binding complex were identified [43][44][45][46] and the corresponding cDNAs were cloned.…”

Section: Tfiiiamentioning

confidence: 99%

Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

et al. 2014

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The discovery of constituents of eukaryotic transcription systems was enabled by the development of in vivo and in vitro methods for their study. Depending on the organism to be studied, the relative importance of each of these two general approaches has been different. Our knowledge about transcription of human genes would be considerably smaller without the results that were obtained by employing in vitro methods. Here, we will mainly focus on in vitro studies of gene expression that contributed to discoveries in the human RNA polymerase (RNAP) III transcription system.In contrast to unicellular yeast, it has until today been and will most likely remain for a many more years impossible to study human RNAP III transcription in cells that have been grown under conditions resembling at least approximately to an in vivo situation in a human being. For that reason, it has hitherto been difficult to address questions concerning the physiological regulation of human RNAP III transcription in its natural environment. However, the identification of the basal components of the RNAP III transcription system could be achieved without directly resorting to a human "in vivo model system." Yet, the identification of human RNAP III subunits and of accessory transcription factors repeatedly took advantage of in vivo systems that were established in other species. Many discoveries of components of the human RNAP III transcription system were fostered by work performed in unicellular (for example, S. cerevisiae and S. pombe) and multicellular eukaryotes (among others, X. laevis or D. melanogaster). Often, results obtained in these model systems helped researchers identify proteins of the human RNAP III transcription system. For instance, the amino acid sequences of transcription factors that were identified and cloned by employing in vivo and in vitro methods in these organisms served as guide for comparing and identifying orthologous transcription factors in human cells (e.g., TFIIIA, TFIIIB [BDP1], TFIIIC35 [GTF3C6]). However, several of the human RNAP III transcription factors were not cloned by homology, but due to extensive purification from human cells (TFIIIB [BRF2], PTF/SNAPc, TFIIIC [GTF3C1-5]). Purification of these factors by employing biochemical methods was only possible, because functional assays were developed that allowed detecting their specific activities. These assays included in vitro transcription, electrophoretic mobility shift assays (EMSA) and footprinting techniques (descriptions of these techniques are found in 1-3 ). Two techniques were essential for the biochemical purification and functional analysis of human transcription factors: (i) the elaboration of protocols that allowed deriving protein extracts from human cells and (ii) the development of cell-free in vitro transcription assays. [4][5][6] Template DNA for in vitro transcription was provided by genes cloned into plasmid DNA. In the case of RNAP III transcription, usually complete and generally short transcribed sequences were included into the...

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“…In vitro transcription was performed in a reconstituted human transcription system, in which a whole cell extract active in RNA polymerase Ill transcription (S100) was depleted of human TFIIIC by phosphocellulose chromatography of HeLa cell extracts (Schneider et al, 1989). This PC-B fraction is transcriptionally inactive.…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

“…HeLa cell extracts can be separated by chromatography into protein fractions containing all factors needed for in vitro transcription except for hTFIIIC (PC-B fraction; Schneider et al, 1989). Such a human protein fraction by itself is inactive in RNA polymerase I11 trancription.…”

Section: Purified B-block-binding Factor H Activates In Vitro Transcrmentioning

confidence: 99%

Isolation of transcription factor IIIC from Dictyostelium discoideum

Bukenberger

Dingermann

Meißner³

et al. 1994

European Journal of Biochemistry

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Transcription factor IIIC (TFIIIC) binds in a sequence-specific manner to RNA-polymerase-IIItranscribed genes (e.g. tRNA genes). It sequesters other transcription factors into the preformed complex, thereby activating transcription by RNA polymerase 111. The Dictyostelium discoideum homologue of TFIIIC was highly purified by af€inity chromatography based on its tDNA-binding activity. This TFIIIC homologue is a multicomponent factor (molecular mass 380 kDa), which binds to the B-box element of the internal tRNA gene promoter without significant A-box interaction. Partially purified D. discoideum TFIIIC is able to functionally complement a human RNA polymerase I11 in vitro transcription system depleted of human TFIIIC. We provide evidence that partially purified D. discoideum TFIIIC interacts in vitro with gene-external B-box elements present downstream of many D. discoideum tRNA genes.DNA-dependent RNA polymerase I11 of eukaryotes catalyses the synthesis of tRNA, 5s rRNA, some small cellular RNAs (e.g. U6 snRNA), and some small viral transcripts (e.g. the adenovirus VA1 RNA; Geiduschek and TocchiniValentini, 1988 ;Willis, 1993). TFIIIC is a universally needed, sequence-specific RNA polymerase I11 transcription factor and is the first protein that binds to the internal promoter sequence of tRNA and viral VA1 genes. The TFIIIC . DNA complex sequesters TFIIIB, a factor that determines RNA polymerase I11 specificity and initiates transcription.TFIIIC has been characterised in considerable detail in several organisms. In all analysed cases, TFIIIC is a multimeric protein complex of at least five proteins (Gabrielsen et al., 1989;Kassavetis et al., 1990;Dean and Berk, 1987;Yoshinaga et al., 1989). A model derived from data using the yeast factor (also called z factor) suggests that TFIIIC may be composed of two functional domains (z, and tB), interacting with the A-block and B-block elements of the internal RNA polymerase I11 promoters (Marzouki et al., 1986;Bartholomew et al., 1990). Affinity-labeling experiments determined that the 138-kDa and 95-kDa subunits of Saccharomyces cerevisiae TFIIIC bind to the B-block and Ablock sequence of the internal promoter, respectively (Bartholomew et al., 1990). Recently, the primary structures of the 138-, 131-and 95-kDa subunits of yeast TFIIIC were reported, deduced from cDNA cloning of the respective genes (Lefebvre et al., 1992). Unlike the yeast factor , human TFIIIC (hTFIIIC) can be separated by chromatography into two domains. TFIIIC2 binds exclu-

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Purification of human transcription factor IIIC and its binding to the gene for ribosomal 5S RNA

Cited by 64 publications

References 39 publications

Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC

Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC

Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

Isolation of transcription factor IIIC from Dictyostelium discoideum

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