Reinigung des Transkriptionsfaktors IIIC1 der RNA-Polymerase III und Identifizierung potentieller TFIIIC1 Untereinheiten

Die humane RNA-Polymerase III synthetisiert kleine, nicht proteinkodierende RNAs, die unterschiedlichen Gengruppen angehören und für deren Herstellung verschiedene Transkriptionsfaktoren benötigt werden. Ein zentraler Transkriptionsfaktor im Polymerase-III-System ist TFIIIC1, der für die Expression...

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Bibliographic Details
Main Author: Hafner, Heike Manuela
Contributors: Prof. Dr. Dr. K. H. Seifart (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2003
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Table of Contents: The human RNA polymerase III produces small, non protein coding RNAs which belong to different gene groups. For transcription of these genes various transcription factors are required. A central transcription factor of the polymerase III transcription system is TFIIIC1 which is essentially required for transcription of all polymerase III depending genes. Neither its structure nor its exact function could be clarified so far. It was investigated whether TFIIIC1 is linked to some proteins effecting polymerase III transcription such as Topoisomerase I and protein kinase CK2. In other experimental systems both of them had shown a positive influence on the efficiency of polymerase III transcription but it could be demonstrated that there is no connection between TFIIIC1 and those proteins. For a better understanding of the exact function of TFIIIC1 as well as for sequencing and cloning of its subunits an advanced purification is required. The Mono S chromatography which was used as new chromatography material lead to a useful purification. Moreover, two different forms of TFIIIC1 could be separated by this chromatography. Different states of modification could be the reason for this chromatographic behaviour. The same phenomenon was observed with the TFIIIC1-like activity which leads to the conclusion that TFIIIC1-like might be a subpopulation of TFIIIC1. Considering also the splitted TFIIIC1 activity, the existence of at least four different forms of TFIIIC1 can be postulated. An additional way to display TFIIIC1 subunits was the assembly of complete and partial VA I transcription complexes which were purified by S500 gelfiltration chromatography (size exclusion chromatography). The isolated partial complexes could be completed by adding the lacking protein fractions and showed a very good transcriptional activity in presence of nucleotides. After comparing the control chromatgraphy runs and the functional TFIIIC1- containing complexes, six polypeptides with sizes of approximately 164, 140, 69, 65, 57 and 43 kDa have been identified to be connected to the TFIIIC1 activity. These sizes could be confirmed by classic chromatography experiments.