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Piaia, Elisa (2005): Molecular function and regulation of the negative cofactor 2, NC2. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Initiation of transcription by eukaryotic RNA polymerase II is finely controlled by a multitude of regulatory factors. Among them, the negative cofactor 2 (NC2), composed of the subunits NC2alpha and NC2beta, is able to bind directly to TBP-DNA complexes, preventing the assembly of the general transcription factors TFIIA and TFIIB. Despite extensive research on the negative and positive function of NC2, several questions concerning its regulation remain unexplored. In particular, localization and post-translational modifications are poorly understood. This work is the first to give some insights on the regulation of this factor. We present evidence that both subunits contain a nuclear localization signal (NLS) responsible for the accumulation of proteins in the nucleus. Immunofluorescence studies showed that NC2 dimer localizes exclusively in the nucleoplasm. However, the two subunits reveal characteristic and unique distribution patterns: NC2alpha is also found in the nucleoli, and NC2beta in small concentrations also in the cytoplasm. Moreover, we show that the two subunits already dimerize in the cytoplasm and are transported into the nucleus as a complex. Interestingly, both NLS are essential for import of the dimer. We also report for the first time several isoforms of both subunits. In vivo labeling experiments showed that NC2alpha is specifically hyperphosphorylated during mitosis. This modification does not impair its ability to dimerize with the partner and bind to TBP-DNA complexes, nor affects the stability of the complex. Furthermore, the phosphorylated protein maintains the ability to mobilize TBP on the DNA. These results suggest that NC2 is still bound to DNA during mitosis, in line with the idea that this factor keeps TBP stably associated to DNA.