umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2001. Vol. 268, no 16, 4527-4536 p.
Keyword [en]
in vitro transcription, RNA polymerase II, ribonucleotide reductase, mouse general transcription factors
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:umu:diva-5476DOI: 10.1046/j.1432-1327.2001.02378.xPubMedID: 11502214OAI: oai:DiVA.org:umu-5476DiVA: diva2:145002
Available from: 2003-09-06 Created: 2003-09-06 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Purification of general RNA polymerase II transcription factors from mouse for studies of proliferation-specific transcription
Open this publication in new window or tab >>Purification of general RNA polymerase II transcription factors from mouse for studies of proliferation-specific transcription
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
Molecular genetics, Genetik
National Category
Genetics
Identifiers
urn:nbn:se:umu:diva-91 (URN)91-7305-500-X (ISBN)
Public defence
2003-09-12, Umeå, 13:00
Opponent
Available from: 2003-09-06 Created: 2003-09-06Bibliographically approved
2. Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation
Open this publication in new window or tab >>Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Deoxyribonucleic acid (DNA) carries all the genetic information of a cell. Ribonucleotide reductase (RNR) provides balanced pools of all four dNTPs, the building blocks of DNA. These building blocks are needed during DNA synthesis and repair. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic abnormalities. Because of its central role in dNTP metabolism, RNR is highly regulated on multiple levels.

The active RNR enzyme consists of two non-identical subunits called proteins R1 and R2. In mammalian cells, during an unperturbed cell cycle, the activity of RNR is highest during S and G2 phases. This is achieved by de novo synthesis of the limiting R2 protein at the onset of S phase, and by controlled degradation of the R2 protein during mitosis.

This thesis deals with both the S phase-specific transcription of the mouse R2 gene, and the M phase-specific degradation of the mouse R2 protein. Sequence comparison of the mouse R2 promoter to human and guinea pig R2 promoters revealed some conserved elements. These putative regulatory elements were tested in both in vitro and in vivo transcription assays. We demonstrated that the previously identified,

NF-Y binding CCAAT box is essential for high-level expression from the R2 promoter, but not for its S phase specificity. In addition, the conserved TATA box is dispensable both for basal and S phase-specific R2 transcription as long as the first 17 basepairs of the 5’ untranslated region are present. However, if this 5’ untranslated region is absent, the TATA box is needed for correct initiation of transcription.

Focusing on the S phase specificity of the R2 gene expression, we demonstrated that the S phase-specific activity of the mouse R2 promoter is dependent on a protein-binding region located ~500 basepairs upstream of the transcription start site and an E2F binding site close to the transcription start site. Deletion of the upstream activating region results in an inactive promoter. In contrast, mutation of the E2F site leads to premature promoter activation in G1 and increased overall promoter activity. However, if the activating mutation of the E2F site is combined with mutation of the upstream activating region, the promoter becomes inactive. These results suggest that the E2F-dependent regulation is important but not sufficient for cell-cycle specific R2 transcription, and that the upstream activating region is crucial for the overall R2 promoter activity.

In our studies of the M phase-specific R2 degradation, we found that it is dependent on a KEN sequence in the N-terminus of the R2 protein, recognized by the Cdh1-APC complex. Mutating the KEN box stabilizes the R2 protein during mitosis and G1 phase.

In summary, these studies further extend our understanding of the regulation of the limiting R2 subunit of the enzyme ribonucleotide reductase. The S phase-specific transcription of the R2 gene and the M phase-specific degradation of the R2 protein may serve as important

mechanisms to protect the cell against unscheduled DNA synthesis.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik, 2003. 34 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 847
Keyword
Molecular biology, ribonucleotide reductase, transcription, protein degradation, cell cycle, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:umu:diva-32 (URN)91-7305-468-2 (ISBN)
Public defence
2003-06-13, KB3A9, KBC-huset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2003-06-04 Created: 2003-06-04 Last updated: 2015-01-19Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Kotova, IrinaChabes, Anna LenaSegerman, BoFlodell, SaraThelander, LarsBjörklund, Stefan
By organisation
Department of Medical Biochemistry and Biophysics
In the same journal
European Journal of Biochemistry
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 100 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf