Transcription by RNA polymerase II requires the assembly of a large preinitiation complex on core promoters of protein-coding genes. A lot of evidence has accumulated in the last few years to suggest that core promoters can be functionally very different. In vitro it has been shown that the affinity with which TBP binds to different core promoters is highly variable and core promoters with weak affinity for TBP (TATA-less) are not transcribed efficiently. This often does not reflect the situation in vivo suggesting that some components essential for transcription of TATA-less core promoters may be missing in vitro. In an attempt to identify such components, we work on five genes (the NOT genes) that have the very interesting and unique property of repressing TATA-less promoters preferentially. Genetic studies suggest that the products of these genes may be associated in a large complex and inhibit transcription by RNA polymerase II at some core promoters selectively, possibly by affecting TFIID function. We have now initiated the purification of Not1p. This protein migrates as 2 separable forms of approximately 200 kDa on SDS gels and can be purified from total yeast extracts in forms that range from 200 kDa to more than 5 MDa. The other Not proteins cofractionate with Not1p through multiple steps of fractionation. We will present the purification of the Not proteins and our attemps to identify the other copurifying factors.