SEARCH · Search PubMed

Results for “TFIIIC”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1989

Human transcription factor IIIC (TFIIIC). Purification, polypeptide structure, and the involvement of thiol groups in specific DNA binding.

Human transcription factor IIIC (TFIIIC) is an initiation factor required for the in vitro transcription of 5 S RNA, tRNA, and adenovirus viral-associated (VA) RNA genes by RNA polymerase III. A TFIIIC activity which complemented purified TFIIIB and RNA polymerase III fractions for VA transcription was highly purified from cultured HeLa cells. This activity copurified through all chromatographic procedures, including B-block oligodeoxynucleotide affinity chromatography, with the two forms of TFIIIC detected by gel mobility shift assays with the VA gene (Hoeffler, W.K., Kovelman, R., and Roeder, R.G. (1988) Cell 53, 907-920). Both specific binding activity to the VAI gene and TFIIIC transcription activity were inhibited by the alkylating agents diisopropyl fluorophosphate, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and N-ethylmaleimide, and to a lesser extent by N alpha-p-tosyl-L-lysine chloromethyl ketone, whereas neither activity was inhibited by phenylmethylsulfonyl fluoride. These data suggest further that the DNA binding and transcription assays scored the same protein(s). TPCK and N-ethylmaleimide inactivated TFIIIC solely through thiol group modification, since prior modification with the reversible thiol reagent 2,2'-dithiopyridine prevented permanent inactivation. The involvement of reduced thiol groups in the specific binding of TFIIIC to the VAI gene was further indicated by an increase in TFIIIC binding activity upon addition of dithiothreitol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mr = 126,000 polypeptide both eluted from a B-block oligodeoxynucleotide affinity column with the DNA binding and transcription activities of TFIIIC and was specifically cross-linked by UV to a 5-bromo-2-deoxynucleotide-substituted B-block oligodeoxynucleotide. The near identity of the TFIIIC molecular weight determined by gel filtration on SOTA Phase GF 200 (Mr = 140,000) suggests that TFIIIC in solution (in the presence of 0.3 M NaCl at pH 7.0) consists of a single polypeptide which is fairly globular in nature.

Adenoviruses, Human↗

PubMed1990

Purification and characterization of Saccharomyces cerevisiae transcription factor TFIIIC. Polypeptide composition defined with polyclonal antibodies.

The class III gene transcription factor termed TFIIIC has been extensively purified from Saccharomyces cerevisiae. Three polypeptides of 138, 131, and 95 kDa consistently copurified with TFIIIC transcription factor activity. These polypeptides were present in approximately equimolar quantities in all TFIIIC preparations. To determine which, if any, of these polypeptides were involved in TFIIIC activity, rabbit polyclonal antibodies were generated against each of these three polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analyses showed that each of the three antiserum preparations reacted uniquely with the respective polypeptide to which they had been elicited. This lack of cross-reactivity by any of the antiserum preparations suggested that these three polypeptides represented distinct unrelated gene products. Each of the three specific antiserum preparations decreased the mobility of TFIIIC-tDNA complexes in a DNA mobility shift assay. More importantly, all three antiserum preparations directly inhibited the transcription factor activity of TFIIIC. In addition, all three antiserum preparations depleted a solution of TFIIIC transcription factor activity. These results indicated that each of these three polypeptides of Mr = 138,000, 131,000, and 95,000 was a distinct and necessary component of yeast TFIIIC. Immunoblot analyses of immunoaffinity-purified TFIIIC fractions indicated that each of the three antiserum preparations alone could deplete the solution of all three polypeptides. These results suggested that these three polypeptides were tightly associated with one another in solution.

Antibodies↗

PubMed1991

A purified adenovirus 289-amino-acid E1A protein activates RNA polymerase III transcription in vitro and alters transcription factor TFIIIC.

We have previously demonstrated that a purified bacterially synthesized E1A 289-amino-acid protein is capable of stimulating transcription from the promoters of genes transcribed by RNA polymerase II in vitro (R. Spangler, M. Bruner, B. Dalie, and M. L. Harter, Science 237:1044-1046, 1987). In this study, we show that this protein is also capable of transactivating in vitro the adenovirus virus-associated (VA1) RNA gene transcribed by RNA polymerase III. Pertinent to the transcription of this gene is the rate-limiting component, TFIIIC, which appears to be of two distinct forms in uninfected HeLa cells. The addition of an oligonucleotide containing a TFIIIC binding site to HeLa whole-cell extracts inhibits VA1 transcription by sequestering TFIIIC. However, the addition of purified E1A to extracts previously challenged with the TFIIIC oligonucleotide restores the level of VA1 transcription. When included in the same reaction, an E1A-specific monoclonal antibody reverses the restoration. Incubation of purified E1A with either HeLa cell nuclear or whole-cell extracts alters the DNA-binding properties of TFIIIC as detected by gel shift assays. This alteration does not occur if E1A-specific antibody and E1A protein are added simultaneously to the extract. In contrast, the addition of this antibody to extracts at a later time does not reverse the alteration observed in the TFIIIC binding activities. Never at any time did we note the formation of novel TFIIIC-promoter complexes after the addition of E1A to nuclear extracts. These results clearly establish that E1A mediates its effect on VA1 transcription through TFIIIC in a very rapid yet indirect manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus Early Proteins↗

PubMed1992

Interaction of Xenopus TFIIIC with the TFIIIA.5 S RNA gene complex.

The general transcription factor TFIIIC is necessary for transcription initiation by RNA polymerase III. TFIIIC binds predominantly to the B-Block promoter element, which is present in tRNA genes, several viral RNA genes and repetitive DNA elements, and to the TFIIIA.DNA complex on 5 S RNA genes. Here we report a characterization of Xenopus laevis TFIIIC and its interaction with the TFIIIA.5 S RNA gene complex. A polypeptide with apparent molecular mass of 85 kDa was specifically cross-linked to a B-Block oligonucleotide by UV light. This polypeptide was present in the partially purified TFIIIC fraction and in a complex with a B-Block double-stranded oligonucleotide isolated by nondenaturing gel electrophoresis. TFIIIC.TFIIIA.DNA gel mobility shift complexes were obtained using B-Block DNA affinity-purified TFIIIC and buffer conditions employing low Mg2+ (1 mM) and high dithiothreitol (7 mM) concentrations. Three TFIIIC.TFIIIA.5 S RNA gene complexes were observed by gel mobility shift analysis. One of these complexes was resistant to dissociation by the addition of competing DNA, but the formation of all three complexes was prevented by the inclusion of excess specific competitor DNA in the initial binding reactions. The apparent affinity of TFIIIC for the TFIIIA.5 S DNA complex was 5-fold higher for the somatic-type 5 S RNA gene than for the oocyte-type 5 S RNA gene. Mutations near the 5' boundary of the TFIIIA binding site alter the DNase I footprint of the TFIIIA.DNA complex and reduce the affinity of TFIIIA-mutant 5 S gene complexes for TFIIIC. Differences in TFIIIC affinity for the two classes of 5 S RNA genes may play a role in the developmental regulation of these gene families.

Animals↗

PubMed2025

Should I stay or should I go: TFIIIC as assembly factor and barrier in RNA polymerase III transcription.

Critical for the regulation of eukaryotic gene transcription is the assembly and interplay of general transcription factors (GTFs) with RNA polymerases (RNAPs), leading to the formation of pre-initiation complexes (PICs) as a rate-limiting step in transcription activation. Compared with RNAPII PIC assembly involving many GTFs, activators, and co-activators, RNAPIII PIC assembly is less complex, involving mainly the four GTFs TFIIIA, TFIIIB, TFIIIC, and snRNA activating protein complex with only a few additional factors. The RNAPIII-specific GTF TFIIIC is present in type I and II promoters. One prominent area of investigation has been the dynamic interaction between TFIIIC and its promoter elements, the varying affinities of TFIIIC toward these elements, and the flexible linker within TFIIIC. Additionally, evidence suggests that TFIIIC may play a dual role, acting as an assembly factor that positions TFIIIB during PIC formation and as a barrier during RNAPIII-mediated transcription. By summarizing recent structural, biochemical, and genomic data, this review explores the mechanisms by which RNAPIII-specific GTFs, with a focus on TFIIIC, dynamically regulate RNAPIII transcription.

RNA Polymerase III↗

PubMed1990

A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells.

In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This inhibition is due primarily to the reduction in transcriptional activity of the pol III transcription factor TFIIIC in poliovirus-infected cells. However, the specific binding of TFIIIC to the VAI gene B-box sequence, as assayed by DNase I footprinting, is not altered by poliovirus infection. We have used gel retardation analysis to analyze TFIIIC-DNA complexes formed in nuclear extracts prepared from mock- and poliovirus-infected cells. In mock-infected cell extracts, two closely migrating TFIIIC-containing complexes, complexes I and II, were detected in the gel retardation assay. The slower migrating complex, complex I, was absent in poliovirus-infected cell extracts, and an increase occurred in the intensity of the faster-migrating complex (complex II). Also, in poliovirus-infected cell extracts, a new, rapidly migrating complex, complex III, was formed. Complex III may have been the result of limited proteolysis of complex I or II. These changes in TFIIIC-containing complexes in poliovirus-infected cell extracts correlated kinetically with the decrease in TFIIIC transcriptional activity. Complexes I, II, and III were chromatographically separated; only complex I was transcriptionally active and specifically restored pol III transcription when added to poliovirus-infected cell extracts. Acid phosphatase treatment partially converted complex I to complex II but did not affect the binding of complex II or III. Dephosphorylation and limited proteolysis of TFIIIC are discussed as possible mechanisms for the inhibition of pol III-mediated transcription by poliovirus.

Acid Phosphatase↗

PubMed1989

Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes.

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.

Binding Sites↗

PubMed1990

Specific interaction of a partially purified Xenopus transcription factor IIIC (TFIIIC) with frog tRNA gene.

Transcription factor IIIC (TFIIIC) from Xenopus has been partially purified and characterized. Footprinting analyses indicate that a partially purified TFIIIC fraction contains an activity which specifically recognizes the "B" block element of tRNA gene. In addition, two other regions located downstream from the "B" block sequence are also protected. Protection experiments on 5S genes by DNAase I with either TFIIIC alone or TFIIIA and TFIIIC produced a minimal change in the cleavage pattern implying that TFIIIC does not intimately associate with DNA. The implications of these findings in relation to the class III gene transcription are discussed.

Animals↗

PubMed1991

Xenopus transcription factor IIIC (TFIIIC) specifically interacts with the "B" block region of the TFIIIA gene.

Transcription factors IIIC (TFIIIC), TFIIIB and RNA polymerase III are commonly required for class III gene transcription in vitro. To understand the diversity and specificity of Xenopus TFIIIC, we have further characterized this factor. Our analyses indicate that a partially purified TFIIIC fraction contains an activity which specifically recognizes the "B" block element of TFIIIA gene. Stable complex formation assays with HeLa cell extracts demonstrate that the TFIIIA gene can stably sequester TFIIIC. off

Animals↗

PubMed1992

Cloning of TFC1, the Saccharomyces cerevisiae gene encoding the 95-kDa subunit of transcription factor TFIIIC.

The yeast gene encoding the 95-kDa subunit of the class III gene transcription factor TFIIIC was cloned. This gene, termed TFC1 (transcription factor C, gene 1), was isolated by screening a lambda gt11 yeast cDNA expression library using a polyclonal antiserum preparation which was previously shown to specifically recognize the 95-kDa subunit of yeast TFIIIC (Parsons, M. C., and Weil, P. A. (1990) J. Biol. Chem. 265, 5095-5103). TFC1 was found to be a single copy gene which contained a continuous open reading frame about 2 kilobases in length. TFC1 was shown to encode the 95-kDa subunit of TFIIIC by several criteria. Like the authentic yeast protein, the protein encoded by TFC1 had an apparent molecular weight of 95,000. In addition, the protein encoded by the TFC1 gene bound to the same antibody species as the yeast 95-kDa subunit of TFIIIC. Last, the sizes of the cleavage products of the Escherichia coli-expressed protein were indistinguishable from those of the cleavage products of the bona fide yeast 95-kDa protein.

Amino Acid Sequence↗

PubMed1988

Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro.

An extract of whole oocytes (oocyte S150) differentially transcribes Xenopus oocyte and somatic 5S RNA genes. In the oocyte S150, transcription complexes with different stabilities are assembled onto oocyte and somatic 5S DNA. The stability of the transcription complex is dependent on activities present in a fraction containing transcription factor TFIIIC. This fraction stabilizes the binding of the positive transcription factor TFIIIA to a somatic 5S RNA gene much more efficiently than it does to an oocyte gene. The oocyte S150 transcription extract is deficient in TFIIIC such that supplementation with a fraction enriched in this transcription factor selectively stimulates oocyte 5S DNA transcription. Previously it has been shown that an egg extract deficient in TFIIIA selectively transcribes somatic 5S RNA genes. Thus under conditions where there is differential stability of transcription complexes, limitation of either TFIIIA or TFIIIC may exaggerate the differential expression of two genes.

Animals↗

PubMed1990

S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors.

The S. cerevisiae RNA polymerase III (pol III) transcription factor TFIIIB binds to DNA upstream of the transcription start site of the SUP4 tRNA(Tyr) gene in a TFIIIC-dependent reaction and to the major 5S rRNA gene in a reaction requiring TFIIIC and TFIIIA. It is shown here that TFIIIB alone correctly positions pol III for repeated cycles of transcription on both genes, with the same efficiency as fully assembled transcription complexes. Thus, TFIIIB is the sole transcription initiation factor of S. cerevisiae pol III; TFIIIC and TFIIIA are assembly factors for TFIIIB. The TFIIIB-dependent binding of pol III to the SUP4 tRNA and 5S rRNA genes has been analyzed in binary (protein and DNA only) and precisely arrested ternary (protein, DNA, and RNA) transcription complexes. Pol III unwinds at least 14 bp of DNA at the SUP4 transcription start in a temperature-dependent process. The unwound DNA segment moves downstream with nascent RNA as a transcription bubble of approximately the same size.

Base Sequence↗

PubMed1991

Binding of yeast TFIIIC to tRNA gene bipartite internal promoters: analysis of physical effects on the intervening DNA.

Complexes between transcription factor TFIIIC and eukaryotic tRNA gene internal promoter A and B boxes are unusual in that the binding to the two distinct sites tolerates considerable variation in both distance and helical orientation between the sites. Electrophoretic mobility of Saccharomyces cerevisiae TFIIIC complexes with circularly permuted tRNA gene fragments and sensitivity of the complexes to a single stranded-specific reagent, potassium permanganate, indicated that no significant bend or distortion was introduced into the DNA by simultaneous binding to both internal promoters. These data support a model in which variability in the relative positions of the two binding sites is compensated by flexibility in the structure of TFIIIC.

DNA↗

PubMed1987

Resolution of human transcription factor TFIIIC into two functional components.

tRNA genes and adenovirus viral-associated (VA) genes are transcribed by RNA polymerase III. Transcription of these genes in vitro requires two protein fractions containing transcription factors designated TFIIIB and TFIIIC, in addition to RNA polymerase III. We report that the TFIIIC fraction derived from human cells in culture can be separated into two functional components, which we call TFIIIC1 and TFIIIC2. Both TFIIIC1 and TFIIIC2 fractions are required for in vitro transcription of the VA1 gene. In DNase I "footprinting" experiments, the TFIIIC2 fraction protects the internal control region termed the B block. Addition of the TFIIIC1 fraction extends the footprint over the internal control region called the A block. TFIIIC1 activity is the limiting transcription factor activity required for VA1 transcription in the crude extract. TFIIIC2 activity sediments as a large component of approximately 18 S, while TFIIIC1 activity sediments at approximately 9 S. These data indicate that the two activities are unique components and when added together reconstitute TFIIIC activity.

Adenoviruses, Human↗

PubMed1987

Two forms of transcription factor TFIIIC in extracts from HeLa cells.

Partially-purified preparations of transcription factor TFIIIC derived from either cytoplasmic (S100) or nuclear (NE) HeLa cell extracts possessed comparable activities when assayed by in vitro transcription, although it was possible to detect sequence-specific binding to the VAI RNA gene promoter by DNase I footprinting only with TFIIIC (NE). On the basis of mixing experiments, this difference could not be ascribed to either transcriptional or DNA-binding inhibitors. These findings provide further evidence for the existence of two forms of TFIIIC: one which is present in both extracts and is transcriptionally active; another which predominates in nuclear extracts and is transcriptionally inactive.

Cell Nucleus↗

PubMed1991

Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC.

A novel photocrosslinking method has been used to identify the components of transcription factor IIIB (TFIIIB) and TFIIIC that associate with DNA upstream of the Saccharomyces cerevisiae SUP4 tRNATyr gene and to map these components to specific positions in DNA. When TFIIIC binds to the tRNA gene, only its second-largest subunit (135 kDa) is accessible for reaction with a photoactive nucleotide, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP, inserted into DNA upstream of the transcriptional start. Formation of TFIII(C + B)-tRNA gene complexes specifically brings two additional polypeptides (90 and 70 kDa) within reach of upstream photoprobes. A collection of 13 probes has been used to map the locations of these three proteins along a 45-bp segment of DNA upstream of the transcriptional start site. Evidence is presented that the 90- and 70-kDa polypeptides are separate and distinct components of yeast TFIIIB, that they are accessible to crosslinking on opposite sides of the DNA helix in a 6-bp segment centered 35 bp upstream of the tRNATyr gene transcriptional start, and that they interact with the second-largest subunit of TFIIIC.

Azides↗

PubMed1992

The herpes simplex virus immediate-early protein ICP27 stimulates the transcription of cellular Alu repeated sequences by increasing the activity of transcription factor TFIIIC.

Infection with herpes simplex virus (HSV) results in an increase in the transcription of the endogenous Alu repeated sequence by RNA polymerase III. This effect is also observed in uninfected cells stably transformed with a plasmid expressing the HSV immediate-early protein ICP27 or in cells transfected with the gene encoding this protein. Both uninfected cells expressing ICP27 and cells infected with virus producing functional ICP27 display increased activity of the cellular transcription factor TFIIIC when compared with untreated cells. This increase is not observed, however, in cells infected with a mutant strain of virus which does not produce ICP27. Hence ICP27 induces elevated Alu transcription by activating transcription factor TFIIIC, which is the limiting factor for such transcription. This is the first report of increased activity of a cellular transcription factor during HSV infection, when most cellular gene activity is inhibited.

Animals↗

PubMed1987

Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay.

Base Sequence↗