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Biochemical and biophysical characterisation of the Saccharomyces cerevisiae cell-cycle transcription factors, SBF and MBF

Pala,, Prashna Jatindra; (2002) Biochemical and biophysical characterisation of the Saccharomyces cerevisiae cell-cycle transcription factors, SBF and MBF. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

MBF and SBF are two large protein complexes involved in cell-cycle-dependent transcriptional regulation in Saccharomyces cerevisiae. These protein complexes bind to DNA sequences called MCBs (Mlu I cell-cycle box) and SCBs (Swi4/Swi6 cell-cycle box) upstream of promoters of many genes that regulate the G1-S phase transition. The binding of MBF to MCBs and SBF to SCBs is highly regulated and requires the association of Mbp1p with Swi6p (MBF), and Swi4p with Swi6p (SBF) through their C-terminal regions. This thesis describes biochemical and biophysical experiments that address crucial protein-protein and protein-DNA interactions involved in SBF and MBF-mediated regulation of the budding yeast cell-cycle. Plasmids expressing various C-terminal fragments of Swi6p, Swi4p, and Mbp1p, containing the heteromerisation regions, have been constructed and their respective proteins purified for structural characterisation. Initially, hydrodynamic and chemical cross-linking data suggested that a C-terminal 18kDa fragment was dimeric. This feature was later shown to be an artefact of disulphide formation in vitro. Mutation of a single conserved cysteine residue within the C-terminus of Swi6p abrogates this dimerisation, but does not show any effect on SBF-mediated transcriptional activation in vivo. The circular dichroic (CD) analyses of the C-terminal Swi6p fragments show a substantial proportion of α-helical secondary structure, verified by comparisons of CD spectra of the full-length molecule and several N- and C-terminally deleted fragments. Limited proteolysis of the 18kDa fragment suggests a bipartite helical domain within the extreme C-terminal region that is required for SBF and MBF interaction. Mutational studies of the DNA-binding region of Mbp1p (1-124) that contains a winged helix-turn-helix motif suggest that the non-conserved residues C-terminal to the core domain are essential for DNA-binding. Mutations of Lys116 (K116A) and Lys122 (K122A) show a reduction in the apparent DNA-binding affinities for the MCB oligonucleotide duplex with respect to the wild-type protein. However, in combination, these two mutations (K116A/K122A) result in a more significant reduction in the apparent binding affinity. The results support the notion of two structurally distinct DNA-binding regions within Mbp1p and related proteins.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Biochemical and biophysical characterisation of the Saccharomyces cerevisiae cell-cycle transcription factors, SBF and MBF
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Transcriptional regulation
URI: https://discovery.ucl.ac.uk/id/eprint/10097625
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