MECHANISM OF TRANSCRIPTION: Studies on the mechanisms controlling transcription initiation and elongation

• Research unit: EU RFP
• Project number: 99.0754

Key words (English)

RNA polymerase II; gene regulation; basal transcription factors; ChIP;
Education; Training; Scientific Research; Social Aspects

Alternative project number (English)

EU project number: HPRN-2000-00087

Research programs (English)

EU-programme: 5. Frame Research Programme - 4.1.1 Research training networks

Short description (English)

See abstract

Further information (English)

Full name of research-institution/enterprise:
Université de Genève
Département de Génétique et Microbiologie
CMU

Partners and International Organizations (English)

MRC (UK); ICRF (UK); FORTH (EL); IGBMC (F)

Abstract (English)

Promoter-assembly of RNA polymerase II transcription complexes in vivo
Promoter-specific activator proteins (activators) function by stimulating formation of RNA polymerase II preinitiation complexes (PICs) at the core promoter of target genes. They are believe to do so by recruiting histone-specific acetylases and/or PIC components to the promoter. A key question, therefore, is how active PICs assemble, and whether this process is the cause or the consequence of histone acetylation, since this has important implications for our understanding of how activators work.
We established a highly quantitative Chromatin Immunoprecipitation (ChIP) assay to assess for increased promoter occupancy by general transcription factors and for changes in histone acetylation in response to natural activators or as a result of tethering specific PIC components to the promoter (see Annual Report 2001). Variations in PIC composition and acetylation states of the histones under these experimental conditions will give us a hint as to how general factors bind or leave the promoter and provide insights into the mode of action of activators. Consistent with published work, we found that the binding of TBP, TFIIB, the Kin28 subunit of TFIIH and RNA Pol II to the GAL1 promoter is strongly increased under inducing conditions, whereas the ADH1 promoter is constitutively occupied. Unfortunately, we failed to detect significant binding of these components to the weak HIS3 promoter that we used in most of our artificial recruitment studies. However, when the HIS core promoter was replaced by the core promoters of other, strongly expressed genes, binding of TFIIB at the activated promoter was readily measurable. Surprisingly, the same increase in promoter occupancy by TFIIB is observed when activation is mediated by the natural VP16 activation domain or by tethering TFIIB to the promoter. We are now in a position to test for promoter occupancy by other general factors and for histone acetylation under these experimental conditions.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 99.0754