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Transcription by RNA-Polymerase III : a paradigm to study eukaryotic gene expression


The objective of this project was to analyze RNA polymerase III (pol-III) and its cognate transcription factors in an attempt to understand the molecular mechanisms underlying the formation of specific transcription complexes on different genes and to gain insight into the regulation of expression patterns of eukaryotic genes in response to external stimuli. We employed the transcription by pol III in different experimental systems (yeast, plant (Arabidopsis) and vertebrate cells) as a paradigm to study DNA-protein and protein-protein interactions among individual transcription factors and with RNA-polymerase III during the formation of the transcription initiation complex. Detailed knowledge of these particular interactions could potentially be important for the development of new techniques to modify by genetic engineering idividual transcription factors involved in the positive or negative regulation of eukarytic gene expression.
Transcription by pol-III involves a multistep assembly of transcription factors into a preinitiation complex which accurately directs initiation by pol III. The factor TFIIIC plays a primary role by binding to the promoter elements of tRNA genes and, once bound, promoters the binding of factor TFIIIB upstream of the transcription start site. TFIIIB-DNA complexes are sufficient to recruit RNA polymerase and to direct multiple rounds of transcription. The subunits of TFIIIC and TFIIIB from yeast cells could be cloned. TFIIIB is a multisubunit protein containing the key iniation factor TBP, which in plant cells binds to the TATA-box in an orientation dependent fashion. It could be shown that components of TFIIIB centrally bridge basal components of the pol-III transcription machinery. In mammalian cells, several factors associate with TBP (so called TAFs) and these polypeptides are thought to play a role both for the regulation and the selectivity by which pol-III is incorporated into particular transcription complexes. In human cells multiple forms of TFIIIB exist, which vary in composition and promoter-specificity. Moreover it could be shown that those pol-III-genes, governed by TATA-containing promoters, (eg. U6-RNA) require a protein complex which primarily binds to the proximal sequence element (PSE) and subsequently directs the formation of the initiation complex. Transcription of pol-III genes can be regulated by activators and a new transcription factor involved in the expression of snRNA genes could be cloned from Xenopus cells (Staf).
1.) Cloning of the subunits of yeast TFIIIB and TFIIIC and the identification of particular protein-protein-contacts as a first step to understand the complicated mechanism by which transcription complexes are specifically formed and regulated.
2. During evolution from yeast to man some pol-III-promoters (eg. U6) have diverged. Accordingly, different forms of the multisubunit complex TFIIIB (TFIIIBalpha and TFIIIbeta) have evolved in human cells which differ in their promoter specificity. Some of the TFIIIB subunits can be mutually exchanged between yeast and man, while others can not. The finding of evolutionary conserved transcription components is potentially an important step to clone human factors by the use of known yeast sequences. In human cells the binding of TFIIIC requires an additional component TFIIIC1, which also binds to the termination site of pol-III genes. This result supports the finding in yeast cells that the integrity of the termination site is important for the process of initiation which is an entirely new observation.
3.) Transcription by pol-III genes is distinctly regulated during various stages of cellular differentiation and proliferation. This can be mediated by modulating the activity of basal factors or by transcriptional activators binding to and operating from upstream sequence elements. The latter finding was hitherto unknown for pol-III genes and reenforces the basic similarity of the nuclear pol-II and pol-III transcription systems.

Funding Scheme

CSC - Cost-sharing contracts


2,Emil-mannkopff-strasse 2
35033 Marburg

Participants (3)

Rue René Descartes 15
67084 Strasbourg
Commissariat à l'Energie Atomique
Centre D'etudes De Saclay
91191 Gif-sur-yvette
66,Maulbeerstrasse 66
4058 Basel