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Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Podcast
Molecular function and regulation of the negative cofactor 2, NC2

Molecular function and regulation of the negative cofactor 2, NC2

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 vor 21 Jahren

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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.