scholarly journals Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

1986 ◽  
Vol 6 (9) ◽  
pp. 3117-3127 ◽  
Author(s):  
M S Kasher ◽  
D Pintel ◽  
D C Ward
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Specific Activity ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Stable Complex ◽  
Polymerase Iii ◽  
S100 Extract ◽  
Affinity Resin ◽  
A Site

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions

1986 ◽  
Vol 6 (9) ◽  
pp. 3117-3127
Author(s):  
M S Kasher ◽  
D Pintel ◽  
D C Ward
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Specific Activity ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Stable Complex ◽  
Polymerase Iii ◽  
S100 Extract ◽  
Affinity Resin ◽  
A Site

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

scholarly journals Stable transcription complex on a class III gene in a minichromosome.

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45 ◽  
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.

Stable transcription complex on a class III gene in a minichromosome

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.

scholarly journals TFIIIC1 acts through a downstream region to stabilize TFIIIC2 binding to RNA polymerase III promoters.

1996 ◽  
Vol 16 (12) ◽  
pp. 6841-6850 ◽  
Author(s):  
Z Wang ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Weak Interaction ◽  
Strong Interaction ◽  
Rna Polymerase Iii ◽  
Cooperative Interaction ◽  
In Vitro System ◽  
Polymerase Iii ◽  
Footprint Analysis

An in vitro system reconstituted with highly purified RNA polymerase III, TFIIIC2, and TFIIIB has been used to identify two chromatographically distinct human RNA polymerase III transcription factors, TFIIIC1 and TFIIIC1', which are functionally equivalent to the previously defined TFIIIC1 (S. T. Yoshinaga, P. A. Boulanger, and A. J. Berk, Proc. Natl. Acad. Sci. USA 84:3585-3589, 1987). Interactions between TFIIIC2, TFIIIC1 (or TFIIIC1'), and the VA1 and tRNA1(Met) templates have been investigated by DNase I footprint analysis. Homogeneous TFIIIC2 alone shows only a weak footprint over the B-box region of the VA1 and tRNA1(Met) templates, whereas TFIIIC1 (or TFIIIC1') alone shows both a strong interaction over the downstream termination region and a very weak interaction near the A-box region. Importantly, when both factors are present simultaneously, TFIIIC1 (or TFIIIC1') dramatically enhances the level of TFIIIC2 binding and extends the footprint to a region that includes the A box. The downstream termination region is essential for this cooperative interaction between TFIIIC2 and TFIIIC1 (or TFIIIC1') on the VA1 and tRNA1(Met) templates and plays a role in the overall accuracy and efficiency of RNA polymerase III transcription.

scholarly journals CK2 Forms a Stable Complex with TFIIIB and Activates RNA Polymerase III Transcription in Human Cells

2002 ◽  
Vol 22 (11) ◽  
pp. 3757-3768 ◽  
Author(s):  
Imogen M. Johnston ◽  
Simon J. Allison ◽  
Jennifer P. Morton ◽  
Laura Schramm ◽  
Pamela H. Scott ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
Rna Polymerase Iii ◽  
Stable Complex ◽  
Class Iii ◽  
Potent Effect ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Chemical Inhibitors

ABSTRACT CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6

1993 ◽  
Vol 13 (6) ◽  
pp. 3231-3244
Author(s):  
B Panning ◽  
J R Smiley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Adenovirus Type ◽  
Open Reading Frames ◽  
Alu Elements ◽  
Polymerase Iii ◽  
Novel Approach ◽  
Adenovirus E1b ◽  
Adenovirus Type 5 ◽  
Reading Frames

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

Analysis of transcription factors binding to the human 7SL RNA gene promoter

1999 ◽  
Vol 77 (5) ◽  
pp. 431-438 ◽  
Author(s):  
Jürgen Müller ◽  
Bernd-Joachim Benecke
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Gene Promoter ◽  
7Sl Rna ◽  
Polymerase Iii ◽  
Responsive Element

Transcription of the human 7SL RNA gene by RNA polymerase III depends on the concerted action of transcription factors binding to the gene-internal and gene-external parts of its promoter. Here, we investigated which transcription factors interact with the human 7SL RNA gene promoter and which are required for transcription of the human 7SL RNA gene. A-box/B-box elements were previously identified in 5S RNA, tRNA, and virus associated RNA genes and are recognized by transcription factor IIIC (TFIIIC). The gene-internal promoter region of the human 7SL RNA gene shows only limited similarity to those elements. Nevertheless, competition experiments and the use of highly enriched factor preparations demonstrate that TFIIIC is required for human 7SL transcription. The gene-external part of the promoter includes an authentic cAMP-responsive element previously identified in various RNA polymerase II promoters. Here we demonstrate that members of the activating transcription factor/cyclic AMP-responsive element binding protein (ATF/CREB) transcription factor family bind specifically to this element in vitro. However, the human 7SL RNA gene is not regulated by cAMP in vivo. Furthermore, in vitro transcription of the gene does not depend on ATF/CREB transcription factors. It rather appears that a transcription factor with DNA-binding characteristics like ATF/CREB proteins but otherwise different properties is required for human 7SL RNA transcription.Key words: 7SL RNA, ATF, CRE, TFIIIC, RNA polymerase III.

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