Characterisation of MBF: A cell cycle regulated fission yeast transcription complex

A key regulatory step in the eukaryotic cell division cycle occurs during the G1 phase. In the case of yeast, cells that are in early G1 may continue through the cell cycle, enter a quiescent state, or undergo sexual differentiation, depending on environmental and nutritional conditions. During a narrow interval in G1 called "START", cells become committed to the option of continuing through the division cycle, and will then do so irrespective of the prevailing conditions. An essential element of the mechanisms that lead to this cell cycle commitment is the transcriptional activation of a subset of cellular genes. In the fission yeast Schizosaccharomyces pombe, this activation is mediated by a transcription factor complex called MBF (also called DSC-1). The overall aim of the work presented in this thesis was to further characterise MBF. Two main experimental approaches were taken to this end. For the first approach, two "One Hybrid" transcription reporter assays were developed to enable transcription factor analysis in fission yeast. These assays were then used to identify a short, conserved, physiologically significant transcriptional activation domain within the Res1 and Res2 components of MBF. The transcriptional activity of Res1 in this assay was found to be Cdc2 independent and not cell cycle regulated. The activity observed from Res2 was found to be dependent on the putative coactivator protein Rep2 which appears to function partly by unmasking the activation domain of Res2. The ankyrin repeat domain of Res2 was found to be important for this activation domain masking. Rep2 was also found to function as a transcriptional activator in the One Hybrid assay, and a sort transcriptional activation domain was mapped to its C-terminus. The transcriptional activity of Rep2 in the One Hybrid assay was also found to be Cdc2 independent, and not cell cycle regulated. For the second experimental approach, "Two Hybrid" screens were performed to isolate cDNAs encoding MBF interacting proteins. A new Res2 interacting protein was identified that is a component of the 26S proteasome