In vitro transcription of a TATA-less promoter: Negative regulation by theNot1 protein

Genetic experiments in the yeast Saccharomyces cerevisiae have identified t he five Not proteins as global repressors of transcription which preferenti ally repress core promoters which do not contain a canonical TATA sequence. Recently, the Ccr4 and Caf1 proteins, required for non-fermentative gene e xpression, were found to be associated with the five Not proteins in 1.2 an d 2 MDa Ccr4-Not complexes. These Ccr4-Not complexes, as many other global regulators of transcription, appear to regulate transcription both positive ly and negatively in vivo. To further characterize the activity of Not1p, the only essential known pro tein of the Ccr4-Not complex, and determine whether it can act directly as a transcriptional repressor, we established an in vitro transcription syste m in which the HIS3 TATA-less promoter can be efficiently transcribed. We d emonstrate that transcription from the HIS3 TATA-less promoter can be speci fically increased in vitro by preparing nuclear extracts from a conditional mutant of the NOT1 gene and analyzing transcription after shifting the nuc lear extracts to the restrictive temperature. This result is the first demo nstration that one of the Not proteins directly represses transcription. Mo reover, it now defines an experimental system in which TATA-less transcript ion initiation and repression by the Ccr4-Not complex can be studied furthe r.