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Purification of general RNA polymerase II transcription factors from mouse for studies of proliferation-specific transcription
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription in vitro, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in E.coli, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II in vitro transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs.

Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of in vivo reporter gene assays and in vitro transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y.

We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and in vitro transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter.

The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an in vitro transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. In vitro transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik , 2003. , 59 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 849
Keyword [en]
Molecular genetics
Keyword [sv]
Genetik
National Category
Genetics
Identifiers
URN: urn:nbn:se:umu:diva-91ISBN: 91-7305-500-X (print)OAI: oai:DiVA.org:umu-91DiVA: diva2:145005
Public defence
2003-09-12, Umeå, 13:00
Opponent
Available from: 2003-09-06 Created: 2003-09-06Bibliographically approved
List of papers
1. A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
Open this publication in new window or tab >>A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
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2001 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 16, 4527-4536 p.Article in journal (Refereed) Published
Abstract [en]

Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.

Keyword
in vitro transcription, RNA polymerase II, ribonucleotide reductase, mouse general transcription factors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-5476 (URN)10.1046/j.1432-1327.2001.02378.x (DOI)11502214 (PubMedID)
Available from: 2003-09-06 Created: 2003-09-06 Last updated: 2011-03-22Bibliographically approved
2. Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
Open this publication in new window or tab >>Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
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2003 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 8, 1791-1801 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAF(II)s. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.

Place, publisher, year, edition, pages
Blackwell Publishing, 2003
Keyword
in vitro transcription, ribonucleotide reductase, TAFs, TATA-box, transcription regulation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-5477 (URN)10.1046/j.1432-1033.2003.03541.x (DOI)000182118800019 ()
Available from: 2003-09-06 Created: 2003-09-06 Last updated: 2015-01-19Bibliographically approved
3. Mechanisms of transcriptional activation of the AdML promoter by NF-Y.
Open this publication in new window or tab >>Mechanisms of transcriptional activation of the AdML promoter by NF-Y.
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5478 (URN)
Available from: 2003-09-06 Created: 2003-09-06 Last updated: 2011-03-22Bibliographically approved

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