Ursula Oberholzer, Martine A. Collart
In Vitro Transcription of a TATA-Less Promoter:
Negative Regulation by the Not1 Protein
Genetic experiments in the yeast Saccharomyces
cerevisiae have identified the five Not proteins as
global repressors of transcription which preferentially
repress core promoters which do not contain a canonical
TATA sequence. Recently, the Ccr4 and Caf1 proteins,
required for non-fermentative gene expression,
were found to be associated with the five Not proteins
in 1.2 and 2 MDa Ccr4-Not complexes. These Ccr4-Not
complexes, as many other global regulators of transcription,
appear to regulate transcription both positively
and negatively in vivo.
To further characterize the activity of Not1p, the only
essential known protein of the Ccr4-Not complex, and
determine whether it can act directly as a transcriptional
repressor, we established an in vitro transcription
system in which the HIS3 TATA-less promoter can
be efficiently transcribed. We demonstrate that transcription
from the HIS3 TATA-less promoter can be
specifically increased in vitro by preparing nuclear extracts
from a conditional mutant of the NOT1 gene and
analyzing transcription after shifting the nuclear extracts
to the restrictive temperature. This result is the
first demonstration that one of the Not proteins directly
represses transcription. Moreover, it now defines an
experimental system in which TATA-less transcription
initiation and repression by the Ccr4-Not complex can
be studied further.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 380, 12/1999
Pages: 1365 - 1370
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