Transcription regulation of the Saccharomyces cerevisiae actin gene

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Authors

Mastrangelo, Peter

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University of Guelph

Abstract

This thesis describes an investigation of how the Saccharomyces cerevisiae actin gene is regulated, with emphasis on the roles of the cellular proteins REB1 and SWI6. The upstream activation sequence (UAS) of the actin gene contains an MluI restriction enzyme site, a hallmark of a DNA element implicated in cell cycle control of gene regulation (the MCB element). This site binds a protein complex, called MBP, which includes Swi6p. Experiments were carried out, using Δswi6 mutant yeast strains, that show that the putative MCB in the actin promoter does not play a role in the cell cycle related transcription exhibited by the actin promoter. The protein REB1, which has binding sites in the promoter of a number of yeast genes, has two sites in the actin UAS. This protein has possible roles as a transcription factor and as a nucleosome organizer. Monoclonal antibodies were made to REB1, and used to show that the products of the SIN3 and SIN4 genes lead to reduced DNA binding by REB1 but do not affect the protein levels, suggesting that DNA binding is somehow regulated in the cell. The ability of REB1 to activate transcription was tested by fusing either the full length protein or fragments of it to a heterologous DNA binding domain, in this case that from the mammalian transcription factor TEF1, and using a LacZ reporter gene having TEF1 binding sites in the promoter. Full length (a.a. 1-809) and truncated (a.a. 205-809) Reb1p retained some transcription activity, while a shorter fragment of Reb1p (a.a. 456-809) repressed transcription. This fragment was found to be able to interact with full length REB1, as measured by gel mobility shift assays. This suggested the possibility that REB1 self-interactions may play a role in the actin UAS, where two REB1 sites occur. This and other data on the DNA binding domains of REB1 are discussed in relation to how the actin promoter might achieve constitutive expression during growth under various conditions.

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Keywords

Saccharomyces cerevisiae actin gene, Regulation, Cellular proteins, Yeast strains, Nucleosome organizer

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