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Structure-function analysis of the MAT[Alpha]2 protein of Saccharomyces cerevisiae Ho, Chi-Yip
Abstract
In Saccharomyces cerevisiae, the MATα2 gene encodes a negative regulatory protein responsible for determining cell-specific mating-type. The MATα2 protein, α2, represses two sets of mating-type-specific genes by interacting with two other factors. α2 forms a heterodimer with the repressor protein a1, and binds a DNA-target site located upstream of all haploid-specific genes, thereby repressing these genes. In combination with a second factor, MCM1, α2 recognizes another DNA-target site found in all the a-specific genes. The binding of α2-MCM1 to these sites leads to the repression of a-specific genes. Therefore, α2 carries out a dual regulatory function in mating-type control through protein-protein interactions with its co- repressors and through the changes in DNA-binding specificities upon interactions with these partners. The objective of this thesis is to gain further insight (i) into the heterodimerization of α2 with al and (ii) into DNA-binding specificity of α2 when acting with MCMI. In studies to localize the important structures within α2 for functional al-α2 heterodimerization, two sequences in the N-terminal half of α2, containing the 3,4-hydrophobic heptad repeat pattern characteristic of coiled-coils, were identified and examined. Amino acid replacements showed that particular positions within the heptads are important in a1-α2 repression and in stabilizing the a1-α2 heterodimer. These mutational analyses are consistent with the proposal that the two heptad sequences of α2 associate with a1 in a coiled-coil-like manner. To determine the DNA-binding specificity of α2 in the presence of MCM1, a variant of the wild-type α2 operator was used as the DNA-target in an in vitro electrophoretic band-shift assay. This variant α2 operator was also used in a heterologous reporter gene system to test the in vivo repression mediated by α2-MCM1. The results together indicate that the nucleotide differences in the variant operator caused a decrease in both α2-MCM1 binding and repression. This suggests that nucleotides outside the core consensus sequence of the α2 operator are important for its in vivo recognition by α2-MCM1.
Item Metadata
Title |
Structure-function analysis of the MAT[Alpha]2 protein of Saccharomyces cerevisiae
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1993
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Description |
In Saccharomyces cerevisiae, the MATα2 gene encodes a negative regulatory protein responsible for
determining cell-specific mating-type. The MATα2 protein, α2, represses two sets of mating-type-specific
genes by interacting with two other factors. α2 forms a heterodimer with the repressor protein a1, and binds
a DNA-target site located upstream of all haploid-specific genes, thereby repressing these genes. In
combination with a second factor, MCM1, α2 recognizes another DNA-target site found in all the a-specific
genes. The binding of α2-MCM1 to these sites leads to the repression of a-specific genes. Therefore, α2
carries out a dual regulatory function in mating-type control through protein-protein interactions with its co-
repressors and through the changes in DNA-binding specificities upon interactions with these partners. The
objective of this thesis is to gain further insight (i) into the heterodimerization of α2 with al and (ii) into
DNA-binding specificity of α2 when acting with MCMI.
In studies to localize the important structures within α2 for functional al-α2 heterodimerization, two
sequences in the N-terminal half of α2, containing the 3,4-hydrophobic heptad repeat pattern characteristic
of coiled-coils, were identified and examined. Amino acid replacements showed that particular positions
within the heptads are important in a1-α2 repression and in stabilizing the a1-α2 heterodimer. These
mutational analyses are consistent with the proposal that the two heptad sequences of α2 associate with a1
in a coiled-coil-like manner.
To determine the DNA-binding specificity of α2 in the presence of MCM1, a variant of the wild-type
α2 operator was used as the DNA-target in an in vitro electrophoretic band-shift assay. This variant α2
operator was also used in a heterologous reporter gene system to test the in vivo repression mediated by
α2-MCM1. The results together indicate that the nucleotide differences in the variant operator caused a
decrease in both α2-MCM1 binding and repression. This suggests that nucleotides outside the core
consensus sequence of the α2 operator are important for its in vivo recognition by α2-MCM1.
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Extent |
4695958 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-04-06
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0088291
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1994-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.