The goal of this research is to elucidate the mechanisms of eucaryotic gene regulation. The system chosen for study is the yeast regulatory complex consisting of CCR4, CAF1, CAF185 and other proteins which are required for the proper expression of a number of diverse genes in yeast. At least five proteins, including CAF1 and CAF185, have been identified as co-immuneprecipitating with CCR4 and another seven appear to co-purify with it. Homologs to CAF1 have been identified in higher organisms, and mammalian CAF1 can substitute for several of yeast CAF1 functions. Biochemical evidence indicates that CCR4 and CAF1 can be specifically retained on a RNA polymerase H C-terminal domain (CTD) affinity column, suggesting that CCR4/CAF1 may be peripheral components of the core transcription apparatus. The CCR4 protein complex is distinct from several other regulatory complexes that have been identified in yeast including that of the SNF/SWI proteins, the holoenzyme SRB complex, the TATA-binding protein associated factors, and the SPT6 complex. The central focus of this grant proposal is two-fold: 1) Identify and characterize the components of the CCR4 protein complex and 2) determine how this complex interacts with the transcriptional machinery to affect transcription. The components of the CCR4 complex will be identified by purifying the complex to homogeneity. The genes for individual components will be cloned following microsequencing of peptide fragments from each protein. The assembly and protein interactions within the complex will be analyzed by genetic and biochemical techniques. The function of CCR4 and its associated proteins will be elaborated by determining their effects on in vitro transcription, by identifying their contacts with the transcriptional machinery, and by examining their link to the CTD of RNA polymerase II. The relationship of CCR4 to that of DBF2, a late mitotic cell-cycle regulated protein kinase that associates genetically with CCR4, will also be studied. Finally, additional regulatory proteins that functionally relate with CCR4 will be identified by characterizing and cloning the genes which when mutated result in synthetic lethality in combination with a ccr4 deletion.
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