The essential function of not1 lies within the Ccr4-not complex¹

Abstract

The five Saccharomyces cerevisiae Not proteins are associated with the Ccr4 and Caf1 proteins in 1.2 MDa and 2 MDa complexes. The Not proteins have been proposed to repress transcription of promoters that do not contain a canonical TATA sequence, while the Ccr4 and Caf1 proteins are required for non-fermentative gene expression. The mechanism of transcriptional regulation by the Ccr4-Not complex is unknown and the role of its different components is unclear. Only Not1p is essential for yeast viability.

Here, we show that most strains carrying combinations of two null alleles of the non-essential CCR4-NOT genes are non-viable. This would suggest that the Ccr4-Not complex is essential. We find that Not1p consists of at least two domains, a C-terminal domain that is essential for yeast viability, and a N-terminal domain that is dispensable but required for yeast wild-type growth. The essential C-terminal domain of Not1p can associate with Not5p, and both proteins are present in 1.2 and 2 MDa complexes in the absence of the N-terminal Not1p domain. In contrast, in the absence of the N-terminal domain of Not1p, Ccr4p does not efficiently associate in large complexes nor with the C-terminal domain of Not1p. Healthy growth is observed when both domains of Not1p are expressed in trans, and is correlated with their physical association, together with Ccr4p, in large complexes. These results are consistent with the essential function of Not1p lying within the Ccr4-Not complex.

Introduction

Accurate transcription by RNA polymerase II requires subunits of RNA polymerase II, basal transcription factors, DNA-binding activators and repressors, and finally, a wide variety of auxiliary factors (reviewed by Hampsey, 1998). These auxiliary factors do not specifically bind DNA but improve or inhibit protein-protein interactions between components of the basal transcription machinery or between the basal transcription machinery and the chromatin template, or between upstream regulators and the basal transcription machinery.

The five NOT genes encode proteins that belong to this category of auxiliary factors Collart and Struhl 1993, Collart and Struhl 1994, Oberholzer and Collart 1998. They were initially isolated in a selection for mutations that increase expression of the HIS3 gene. Mutations in these NOT genes were found to have the unique characteristic of preferentially increasing transcription from the so-called TATA-less promoter of the HIS3 gene. They also increase transcription of a large number of unrelated genes, suggesting that the NOT genes encode global negative regulators of transcription. This conclusion is supported by the recent observation that not mutations can suppress the temperature sensitivity associated with a mutation in SRB4, which encodes a component of the RNA polymerase II holoenzyme required for the transcription of most genes (Lee et al., 1998). Furthermore, the transcriptional activity resulting from a functional Spt3p-TBP interaction (Eisenmann et al., 1992) has been reported to be repressed by the Not1 protein (Collart, 1996). Because they can distinguish between core promoters, it has been suggested that the Not proteins regulate TFIID activity (Collart & Struhl, 1994).

Genetic and biochemical experiments have suggested that the products encoded by the NOT genes are associated in a large complex Collart and Struhl 1994, Oberholzer and Collart 1998. Recently, a 1.2 MDa multiprotein complex containing Ccr4p, Caf1p and the five Not proteins has been described (Liu et al., 1998). CCR4 was identified as a gene required for maximal induction of ADH2 transcription under non-fermentative growth conditions. It is required for full derepression of many non-fermentative genes under glucose-derepressed conditions, but also appears to play a transcriptional role in diverse cellular events Denis 1984, Denis and Malvar 1990. The Caf1/Pop2 protein interacts with Ccr4p (Draper et al., 1995) and caf1 disruptions induce phenotypes and transcriptional defects very similar to those observed with ccr4. Ccr4p, Caf1p and the five Not proteins also co-fractionate with a size of approximately 2 MDa (Bai et al., 1999, and our unpublished observations).

The Ccr4-Not complex is a global regulator of transcription that regulates genes both positively and negatively. Indeed, the NOT genes have been defined as global negative regulators of transcription because recessive and loss of function mutations lead to an increase in transcription of a number of genes. The CCR4 and CAF1 genes have been defined mostly as positive transcription factors. Only a few of the phenotypes associated with mutations in the CCR4 and CAF1 genes are also associated with mutations in some of the NOT genes and vice versa (Liu et al., 1998, and our unpublished observations). In fact, we find that mutations in CAF1 and CCR4 do not lead to increased HIS3 expression as do mutations in NOT genes, and moreover suppress this phenotype in not mutants (unpublished observations). Recently, it has been shown that these functional differences can be correlated with the association of Caf1p and Ccr4p on one hand, and Not2p, Not4p and Not5p on the other hand, to different regions of Not1p (Bai et al., 1999).

Here, we have investigated the function of Not1p within the Ccr4-Not complex. We observed that nearly any combination of two ccr4-not null mutations is lethal, as is the deletion of the NOT1 gene alone. We were able to define at least two domains of Not1p that can functionally and physically interact when expressed in trans: an essential C-terminal domain, and a N-terminal domain that is required for wild-type growth. We could show that efficient association of Ccr4p with the C-terminal domain of Not1p required the N-terminal domain of Not1p. Our results suggest that the essential function of Not1p lies within the Ccr4-Not complex.