Transcription factor interactions at the PGK promoter in yeast

Packham, Elizabeth A. (1996) Transcription factor interactions at the PGK promoter in yeast. PhD thesis, University of Nottingham.

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Abstract

Two new transcription factor binding sites have been identified within the phosphoglycerate kinase (PGK) gene promoter in the yeast Saccharomyces cerevisiae. The binding sites are upstream of the previously defined UAS, and are bound in vitro by the multifunctional transcription factors Reb1p and Cpf1p. A deletion of the Reb1p binding site was made from a PGK gene construct on a multicopy plasmid, and also targeted to the chromosomal copy of PGK. Deletions of the Rap1p and Abf1p binding sites in the UAS were also targeted to the chromosome. Analysis of RNA from the chromosomal deletion strains confirmed the central role of Rap1p in the activation of transcription from PGK. Reb1p and Abf1p were also found to be important for transcriptional activation. This is in contrast to results from experiments using multicopy plasmids carrying Reb1p or Abf1p binding site deletions from PGK. In this situation, neither the Reb1p site, nor the Abf1p site, plays a role in transcriptional activation.

A role for Cpflp at the PGK promoter was examined using a cpfl null strain of yeast. Northern blot analysis was used to assay transcription from the chromosomal PGK gene in the absence of Cpf1p, and also transcription from a multicopy plasmid carrying the wild type PGK gene in the cpf1- background. In both cases, the absence of Cpf1p was found to have very little effect on the level of transcription.

In addition, a role for the potential yATF binding site at the 3' end of the PGK UAS was investigated. Oligonucleotides containing this sequence were inserted upstream of a minimal promoter, and levels of a β-galactosidase reporter were assayed. No activation over the basal level was observed. A deletion of the potential yATF binding site from the UAS was made from a multicopy plasmid construct, and also from the chromosomal locus. Transcription from the deleted constructs was found to be no different from transcription from the wild type gene.

Finally, DNA sequences which are able to complement the C-terminus functions of Rap1p were identified. A yeast genomic library was generated downstream of the N-terminus and DNA binding domain of Rap1p. This library was transformed into a rap1ts strain of yeast to look for complementation of the ts phenotype. Transformants which grew at the non-permissive temperature were obtained. Results from the analysis of the DNA sequences in these transformants are presented.

Item Type:Thesis (PhD)
Supervisors:Chambers, A.
Uncontrolled Keywords:genetics, molecular biology, transcription factors, binding sites, Saccharomyces cerevisiae, PGK gene promoter
Faculties/Schools:UK Campuses > Faculty of Science > School of Biosciences
ID Code:1575
Deposited By:Ms. K EVANS
Deposited On:12 Oct 2010 11:54
Last Modified:12 Oct 2010 11:54

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