The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively

The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co‐immunoprecipitate with CCR4 and CAF1, and NOT1 co‐purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2×106 Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1–lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

[1]  C. Denis,et al.  Factors Affecting Saccharomyces cerevisiae ADH2Chromatin Remodeling and Transcription* , 1997, The Journal of Biological Chemistry.

[2]  R. Young,et al.  RNA Polymerase II Holoenzyme Contains SWI/SNF Regulators Involved in Chromatin Remodeling , 1996, Cell.

[3]  M. Mann,et al.  Cell biology and the genome projects a concerted strategy for characterizing multiprotein complexes by using mass spectrometry. , 1997, Trends in cell biology.

[4]  T. Chibazakura,et al.  Molecular analysis of POP2 gene, a gene required for glucose-derepression of gene expression in Saccharomyces cerevisiae. , 1992, Nucleic acids research.

[5]  M. Collart.  The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters , 1996, Molecular and cellular biology.

[6]  L. Johnston,et al.  The cell-cycle-regulated budding yeast gene DBF2, encoding a putative protein kinase, has a homologue that is not under cell-cycle control. , 1991, Gene.

[7]  C. Denis,et al.  Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex , 1995, Molecular and cellular biology.

[8]  G. Natsoulis,et al.  The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci. , 1991, The New biologist.

[9]  D. Schild,et al.  Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. , 1995, Genetics.

[10]  K. Irie,et al.  The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes , 1994, Molecular and cellular biology.

[11]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[12]  R. Hawley,et al.  The effect of mei-41 on rDNA redundancy in Drosophila melanogaster. , 1983, Genetics.

[13]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  W. Cook,et al.  Dissection of the ADR1 protein reveals multiple, functionally redundant activation domains interspersed with inhibitory regions: evidence for a repressor binding to the ADR1c region , 1994, Molecular and cellular biology.

[15]  H. Mitsui,et al.  Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae. , 1998, Genetics.

[16]  K. Struhl,et al.  CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters. , 1993, The EMBO journal.

[17]  C. Denis.  Identification of new genes involved in the regulation of yeast alcohol dehydrogenase II. , 1984, Genetics.

[18]  K. Struhl,et al.  NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization. , 1994, Genes & development.

[19]  M. Wilm,et al.  Error-tolerant identification of peptides in sequence databases by peptide sequence tags. , 1994, Analytical chemistry.

[20]  A. Podtelejnikov,et al.  Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. , 1996, Rapid communications in mass spectrometry : RCM.

[21]  L. Johnston,et al.  DBF2, a cell cycle‐regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex , 1997, The EMBO journal.

[22]  T. Malvar,et al.  The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression. , 1990, Genetics.

[23]  H. Y. Liu,et al.  CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context , 1994, Molecular and cellular biology.

[24]  L. Johnston,et al.  Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. , 1996, The EMBO journal.

[25]  D. E. Levin,et al.  Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect , 1992, The Journal of cell biology.

[26]  R Ohba,et al.  Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. , 1997, Genes & development.

[27]  B. Errede,et al.  MOT2 encodes a negative regulator of gene expression that affects basal expression of pheromone-responsive genes in Saccharomyces cerevisiae , 1994, Molecular and cellular biology.

[28]  Kim Nasmyth,et al.  Cell cycle control of the yeast HO gene: Cis- and Trans-acting regulators , 1987, Cell.

[29]  H. Y. Liu,et al.  The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and forms a functionally distinct complex from that of the SNF/SWI transcription factors. , 1994, Genetics.

[30]  M. Mann,et al.  Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Wilm,et al.  Analytical properties of the nanoelectrospray ion source. , 1996, Analytical chemistry.

[32]  L. Guarente,et al.  Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site , 1983, Cell.

[33]  M. Collart,et al.  Characterization of NOT5 that encodes a new component of the Not protein complex. , 1998, Gene.

[34]  N. Thompson,et al.  A novel collection of accessory factors associated with yeast RNA polymerase II. , 1996, Protein expression and purification.