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PubMed1997

RNA polymerase III transcription repressed by Rb through its interactions with TFIIIB and TFIIIC2.

The retinoblastoma susceptibility gene product (Rb) generally represses RNA polymerase III (Pol III)-directed transcription. This implies that Rb interacts with essential transcription factors. Mutations in either the A or B subdomains in the Rb pocket interfere with Rb-mediated repression of Pol III-directed transcription, which indicates that both subdomains are directly involved in this activity. Addition of either purified TFIIIB or purified TFIIIC2 partially relieves Rb-mediated repression and restores activity to nuclear extracts that had been depleted of essential factors by binding to Rb. Pull down and coimmunoprecipitation experiments as well as functional assays indicate that Rb interacts with both TFIIIB and TFIIIC2 and that the A subdomain is primarily required for binding TFIIIB and the B subdomain for binding TFIIIC2. While Rb interacts with both factors, the A subdomain is more important than the B subdomain in directing Rb-mediated repression, and TFIIIB is the principal target of that activity.

Amino Acid Sequence↗
PubMed2001

The activity of transcription factor IIIC1 is impaired during differentiation of F9 cells.

Differentiation in vitro of mouse F9 embryonal carcinoma (EC) cells to the parietal endoderm (PE) mimics processes of development of the early mouse embryo. This differentiation is accompanied by a dramatic down-regulation of all genes transcribed by RNA polymerase III (pol III). Complementation of extracts from cells, differentiated for various time periods with purified pol III transcription factors show for the first time that TFIIIC1 can substantially restore this impaired transcription, particularly in the early stages of differentiation. At later stages (day 7) the TBP (TATA-binding protein )-TAF complex, TFIIIBbeta, may also become limiting, which can contribute to but cannot account for the reduced transcription of type 2 promoters in PE cells. Because TFIIIBbeta is not required for the expression of type 3 promoters, other components must necessarily be involved, and our results show that U6 transcription can significantly be reactivated by TFIIIC1. By employing a variant type 3 promoter construct, which essentially requires a mutant form of TBP (TBP-DR2), we show that TBP is not limiting in PE extracts. The partial purification of pol III transcription factors from PE and EC cells revealed that TFIIIC2 activity could be purified from both cell types, whereas TFIIIC1 activity was dramatically reduced in extracts from PE cells.

Animals↗
PubMed2004

Transcription factor (TF)-like nuclear regulator, the 250-kDa form of Homo sapiens TFIIIB", is an essential component of human TFIIIC1 activity.

The general human RNA polymerase III transcription factor (TF) IIIC1 has hitherto been ill defined with respect to the polypeptides required for reconstitution of its activity. Here we identify Homo sapiens TFIIIB" (HsBdp1) as an essential component of hTFIIIC1 and hTFIIIC1-like activities. Several forms of HsBdp1 are described. The 250-kDa form of HsBdp1, also designated the "transcription factor-like nuclear regulator," strictly co-eluted with TFIIIC1 activity over multiple chromatographic purification steps as revealed by Western blot with anti-HsBdp1 antibodies and by MALDI-TOF analysis. In addition, TFIIIC1 activity could be depleted from partially purified fractions with anti-HsBdp1 antibodies but not with control antibodies. Moreover, highly purified recombinant HsBdp1 could replace TFIIIC1 activity in reconstituted transcription of the VAI gene in vitro. Furthermore, smaller proteins of approximately 90-150 kDa that were recognized by anti-HsBdp1 antibodies co-eluted with TFIIIC1-like activity. Finally, cytoplasmic extracts from differentiated mouse F9 fibroblast cells that lacked TFIIIC1 activity could be made competent for transcription of the VA1 gene by the addition of TFIIIC1, TFIIIC1-like, or recombinant HsBdp1. These results suggest that HsBdp1 proteins represent essential components of TFIIIC1 and TFIIIC1-like activities.

Adenoviridae↗
PubMed1997

Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein.

The tumour suppressor protein RB restricts cellular growth. This may involve inhibiting the synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III. We have shown previously that RB can repress pol III transcription when overexpressed either in vitro or in vivo. We also demonstrated that pol III activity is elevated substantially in primary fibroblasts from RB-deficient mice. Here we address the molecular mechanism of this regulation. RB is shown to repress all types of pol III promoter. It can do this even if added after transcription complex assembly. Functional assays demonstrate that RB targets specifically the general pol III factor TFIIIB. A physical interaction between TFIIIB and RB is indicated by fractionation, pull-down and immunoprecipitation data. We show that TFIIIB activity is elevated in primary fibroblasts from RB-deficient mice. TFIIIB is a multisubunit complex that includes the TATA-binding protein (TBP) and a TFIIB-related factor called BRF. We show that RB itself contains regions of homology to both TBP and BRF and propose a model in which RB disrupts TFIIIB by mimicking these two components.

Amino Acid Sequence↗
PubMed2003

p53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB.

The tumor suppressor p53 is a transcription factor that controls cellular growth and proliferation. p53 targets include RNA polymerase (pol) III-dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA-binding protein (TBP)-containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP-p53 interaction is of functional importance for regulating RNA pol III-transcribed genes. Unlike RNA pol II-dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB.

Animals↗
PubMed1989

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

Transcription factor hTFIIIC was purified from cytoplasmic extracts of HeLa cells using four different chromatographic steps. This procedure yields a protein fraction which actively supports transcription in reconstitution assays and contains five major polypeptide chains with a molecular mass ranging from 25 to 250 kDa as estimated by SDS-PAGE and silver staining. In this fraction a polypeptide with a molecular mass of approximately 110 kDa could be identified as a specific DNA-binding component of hTFIIIC. By electrophoretic mobility shift and footprinting analyses it could be demonstrated that purified hTFIIIC binds specifically to the 5S gene. The protected region encompasses the A-Box promoter element and flanking sequences extending toward the 5'-proximal end of the gene. By addition of hTFIIIC to preformed TFIIIA/5S DNA complexes, we observe an additive effect of both factors on the footprint boundaries.

Animals↗
PubMed1989

Magnetic DNA affinity purification of yeast transcription factor tau--a new purification principle for the ultrarapid isolation of near homogeneous factor.

We present a new method for rapid purification to near homogeneity of sequence specific DNA binding proteins based on magnetic separation. The method is described for the purification of the yeast transcription factor tau. DNA affinity Dynabeads (monodisperse superparamagnetic particles) specifically bind the protein in the presence of competitor DNA. By magnetic separation, wash and elution, highly enriched transcription factor preparations are obtained within minutes. In less than an hour with three cycles of adsorption, nearly homogeneous factor tau was obtained. The factor preparation contained mainly two polypeptides of 100 and 140 kDa and was fully active in transcription and DNA binding assays. This procedure should work for any high-affinity sequence-specific DNA binding protein with only minor modifications.

Adsorption↗
PubMed1989

A DNA-binding domain of human transcription factor IIIC2.

Transcription factor IIIC2 is required for in vitro transcription of the adenovirus 2 VA1 gene and binds with high affinity to its B-box promoter element which is an 18 bp perfect inverted repeat. Partial proteolysis of TFIIIC2 with chymotrypsin and Staphylococcus aureus V8 protease yielded a species which produced a discrete band in a gel shift assay with about twice the mobility of the undigested complex. Chymotrypsin-digested TFIIIC2 produced a DNase I footprint virtually identical to that of the undigested protein, but the stability of the protein-VA1 DNA complex was drastically reduced and the in vitro transcriptional activity was eliminated. These results indicate that a chymotrypsin-resistant domain of TFIIIC2 binds to the B-box sequence. We speculate that stable binding requires protease sensitive cooperative interactions between TFIIIC2 DNA-binding domains.

Adenoviruses, Human↗
PubMed1990

Human transcription factor IIIC contains a polypeptide of 55 kDa specifically binding to Pol III genes.

Human transcription factor IIIC contains a 55 kDa polypeptide which specifically interacts with the Adenovirus 2 VAI gene promoter and which mimics most of the DNA binding properties of the entire factor. The specificity and affinity of this protein:DNA interaction was demonstrated by: (i) Separation of purified fractions of hTFIIIC by SDS PAGE, electrotransfer to nitrocellulose, renaturation of proteins and their subsequent binding to the VAI gene, (ii) recovery and renaturation of proteins from SDS gels and identification of a fraction of hTFIIIC with a molecular mass less than 68 kDa, which specifically binds to VAI DNA, (iii) correlating the differential binding activity of the renatured 55 kDa component of hTFIIIC to mutated Pol III promoters with the ability of the entire factor to form functional transcription complexes thereon, and finally by (iv) specific crosslinking of the 55 kDa DNA binding component of hTFIIIC to the photoaffinity labeled B-box promoter sequence of the VAI gene.

Adenoviridae↗
PubMed1991

Yeast TFIIIA + TFIIIC/tau-factor, but not yeast TFIIIA alone, interacts with the Xenopus 5S rRNA gene.

The successful use of mixed heterologous in vitro transcription systems has suggested that the species specificity of RNA polymerase III transcription is low. To see if this extends to lower eukaryotic class III transcription factors, we compared the interactions of the two yeast assembly factors, TFIIIA and TFIIIC/tau factor, with a homologous yeast 5S rRNA gene and a heterologous Xenopus laevis somatic 5S rRNA gene. Transcription assays showed that the Xenopus gene was transcriptionally inactive in a crude cell-free yeast extract that actively transcribes the homologous gene. However, the Xenopus gene was still able to compete for limiting transcription factors. Electrophoretic DNA binding assays revealed that while TFIIIA bound avidly to the yeast gene (generating the 'A-complex'), it had no affinity for the Xenopus 5S rRNA gene. Nevertheless, a complex of both TFIIIA and TFIIIC/tau factor (the 'AC-complex') was formed on the two genes with similar affinity, although only the complex assembled on the homologous gene was able to activate transcription. Thus enough sequence information is present on the heterologous gene to direct transcription factor assembly, but not to activate transcription. Like its counterpart in Xenopus, the yeast TFIIIA appears to be a zinc binding protein that is inactivated by EDTA and 1,10-phenanthroline, and reactivated in the presence of zinc ions. Bound to the 5S rRNA gene, TFIIIA is however significantly more resistant to inactivation by chelators than in its free state. The AC-complex differs from the A-complex by being less affected by chelators, and by being more sensitive to the dissociating effect of single-stranded DNA.

Animals↗
PubMed1991

Human transcription factor IIIC binds to its cognate promoter sequences in a metal coordinated fashion.

Transcription factor IIIC from human cells (hTFIIIC) contains a 55 kDa polypeptide which specifically binds to the promoter of the VAI and 5S gene. This interaction can be abolished by depleting divalent metal cations from the free protein through chelation with EDTA. Prior association of the protein with its DNA-binding sequence renders the complex refractory to chelation by EDTA. Specific binding of hTFIIIC to its cognate promoter sequences--shown by electrophoretic mobility shift and DNase I protection assays--can be restored by the addition of zinc ions. In contrast to the binding of hTFIIIA to the 5S gene, which was monitored in parallel and which exclusively requires Zn2+, the binding of hTFIIIC to the VAI and 5S gene can also be reconstituted--albeit with a lower efficiency--by the transition metals Co2+, Fe2+ and Mn2+ but not by Ni2+ or Cu2+. These results show that hTFIIIC binds to its promoter sequences in a metal coordinated fashion which differs from that observed for the binding of hTFIIIA to the 5S gene.

Cations, Divalent↗
PubMed1993

Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.

A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.

Adenoviridae↗
PubMed1993

Transcription factor IIA stimulates the expression of classical polIII-genes.

Protein fractions containing TFIIA, a transcription factor known to be involved in transcription initiation by RNA polymerase II and 5'-regulated polymerase III genes (e.g. U6), were tested for their role in in vitro transcription of classical pol III genes. These fractions were shown to stimulate a basal transcription system, reconstituted from highly purified fractions hTFIIIB and hTFIIIC. We demonstrate that this stimulating activity isolated from HeLa cells coelutes over at least six chromatographic steps with hTFIIA. Moreover the native molecular mass and the stability of this activity against heat treatment are comparable to those of hTFIIA. Finally we show that recombinant TFIIA from Saccharomyces cerevisiae can substitute for the human factor in pol III transcription in vitro which proves that TFIIA is also involved in the efficient expression of classical pol III genes.

Humans↗
PubMed1997

Human transcription factors IIIC2 , IIIC1 and a novel component IIIC0 fulfil different aspects of DNA binding to various pol III genes.

Human transcription factor IIIC2 interacts with the TFIIIA-5S DNA complex and forms a ternary TFIIIA/IIIC2-5S DNA complex. Formation of this complex does not preclude simultaneous binding of TFIIIC2to the B-box sequence of a second template. This suggests that the domain(s) or subunit(s) required for indirect recognition of the 5S promoter by TFIIIC2 are different from those necessary for direct binding of TFIIIC2 to B-box-containing pol III promoters. Whereas TFIIIC2 is only required for transcription of the 'classical' pol III genes, TFIIIC1 is generally required for transcription of all pol III genes, including that of the U6 gene. The activity of TFIIIC1 strongly enhances specific binding of basal pol III factors TFIIIA, TFIIIC2 and the PSE binding protein (PBP) to their cognate promoter elements and it acts independently of the corresponding termination regions. Moreover, we characterize an activity, TFIIIC0, purified from phosphocellulose fraction C, which shows strong DNase I protection of the termination region of several pol III genes and which is functionally and chromatographically distinct from TFIIIC1 and TFIIIC2.

Animals↗
PubMed1998

The activity binding to the termination region of several pol III genes represents a separate entity and is distinct from a novel component enhancing U6 snRNA transcription.

Human TFIIIC1, a basal transcription factor essentially required for expression of all pol III genes, exerts its function without primarily binding to DNA. We report here the purification of a termination site binding activity (TBA) which was initially described to be contained in fractions designated as TFIIIC0. TBA specifically and strongly binds to the termination region of pol III genes with internal promoters and can be completely separated from TFIIIC1and a TFIIIC1related activity (TFIIIC1-like), proving that DNA-binding of TBA is independent of these latter activites. Although TBA is not essentially required for, it strongly stimulates pol III transcription from intragenic promoters. This stimulation strictly depends on the presence of TFIIIC1and is not observed in conjunction with TFIIIC1-like. We further present the identification of a novel activity, TFIIIU, which is also contained in crude fractions of TFIIIC0. TFIIIU can be separated from TBA by further purification and is essentially involved in transcription of the mammalian U6 gene. TFIIIU cannot be substituted for by any of the established U6 transcription factors and thus represents a novel U6 transcription factor.

Binding Sites↗
PubMed2001

Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein.

We attempted to devise a transcription system in which a particular DNA sequence of interest could be inducibly expressed under the control of a modified polymerase III (pol III) promoter. Its activation requires a mutated transcription factor not contained endogenously in human cells. We constructed such a promoter by fusing elements of the beta-lactamase gene of Escherichia coli, containing a modified TATA-box and a pol III terminator, to the initiation region of the human U6 gene. This construct functionally resembles a 5'-regulated pol III gene and its transcribed segment can be exchanged for an arbitrary sequence. Its transcription in vitro by pol III requires the same factors as the U6 gene with the major exception that the modified TATA-box of this construct only interacts with a TATA-binding protein (TBP) mutant (TBP-DR2) but not with TBP wild-type (TBPwt). Its transcription therefore requires TBP-DR2 exclusively instead of TBPWT: In order to render the system inducible, we fused the gene coding for TBP-DR2 to a tetracycline control element and stably transfected this new construct into HeLa cells. Induction of such a stable and viable clone with tetracycline resulted in the expression of functional TBP-DR2. This system may conceptually be used in the future to inducibly express an arbitrary DNA sequence in vivo under the control of the above mentioned promoter.

Base Sequence↗
PubMed2003

Assembly and isolation of intermediate steps of transcription complexes formed on the human 5S rRNA gene.

By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene. These large complexes were separated by size exclusion chromatography from non- incorporated proteins. In addition, we succeeded in isolating specific intermediate stages of complex formation. Such isolated partial complexes require complementation with the missing activities for full transcription activity. One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1. Thus TFIIIC1 is not an assembly factor for other transcription factors. Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity. This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III. These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.

Cell Line↗
PubMed1997

Casein kinase II regulation of yeast TFIIIB is mediated by the TATA-binding protein.

The highly conserved protein kinase casein kinase II (CKII) is required for efficient Pol III transcription of the tRNA and 5S rRNA genes in Saccharomyces cerevisiae. Using purified factors from wild-type cells to complement transcription extracts from a conditional lethal mutant of CKII we show that TFIIIB is the CKII-responsive component of the Pol III transcription machinery. Dephosphorylation of TFIIIB eliminated its ability to complement CKII-depleted extract, and a single TFIIIB subunit, the TATA-binding protein (TBP), is a preferred substrate of CKII in vitro. Recombinant TBP purified from Escherichia coli is phosphorylated efficiently by CKII and, in the presence of a limiting amount of CKII, is able to substantially rescue transcription in CKII-deficient extract. Our results establish that TBP is a key component of the pathway linking CKII activity and Pol III transcription and suggest that TBP is the target of a CKII-mediated regulatory mechanism that can modulate Pol III transcription.

Casein Kinase II↗