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Structural and thermodynamic studies...

Ke, Ailong.

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Structural and thermodynamic studies of the DNA binding properties of yeast MATalpha2 and MATa1 proteins.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Structural and thermodynamic studies of the DNA binding properties of yeast MATalpha2 and MATa1 proteins./
作者:	Ke, Ailong.
面頁冊數:	102 p.
附註:	Adviser: Cynthia Wolberger.
Contained By:	Dissertation Abstracts International63-03B.
標題:	Biology, Molecular. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3046480
ISBN:	049360670X

Structural and thermodynamic studies of the DNA binding properties of yeast MATalpha2 and MATa1 proteins.
Ke, Ailong.

Structural and thermodynamic studies of the DNA binding properties of yeast MATalpha2 and MATa1 proteins. - 102 p.

Adviser: Cynthia Wolberger.

Thesis (Ph.D.)--The Johns Hopkins University, 2002.

In diploid yeast cells, the MATα2 and MAT<bold>a</bold>1 homeodomain proteins bind DNA cooperatively to repress transcription of haploid-specific genes. In haploid α cells, MATα2 binds DNA cooperatively with MCM1 to repress <bold>a</bold>-specific gene (<italic>asg</italic>) transcription. Although the α2 protein binds DNA in a similar manner as other homeodomain proteins, a triple alanine mutant, in which the DNA major groove-contacting side chains are mutated to alanines, shows context-dependent DNA binding <italic> in vitro</italic> and <italic>in vivo</italic>. This triple mutant, called α2-3A, does not binds DNA detectably by itself or in complex with MCM1. Paradoxically, α2-3A still binds DNA in complex with <bold>a</bold>1 with unreduced affinity and specificity. To resolve this paradox, I determined the crystal structure of the <bold>a</bold>1/α2-3A heterodimer bound to DNA. The structure shows that the triple mutation causes a collapse of the α2-3A/DNA interface, giving rise to a re-organized set of contacts between α2-3A and DNA, that accounts for the ability of α2-3A and <bold>a</bold>1 to recognize the wild type DNA sequence. Isothermal titration calorimetry experiments revealed that a much more favorable entropic component stabilizes the <bold>a</bold>1/α2-3A/DNA complex, as compared with the α2-3A/DNA complex. These results suggest that either some of the reorganized α2-3A/DNA interactions are missing when α2-3A binds DNA alone, or that the <bold>a</bold>1/α2-3A/DNA complex is much more flexible than the wild type complex. The combined structural and thermodynamic study provides an explanation for how partner proteins can influence the sequence specificity of a DNA binding protein.

ISBN: 049360670XSubjects--Topical Terms:

1017719
Biology, Molecular.

Structural and thermodynamic studies of the DNA binding properties of yeast MATalpha2 and MATa1 proteins.
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502 $a Thesis (Ph.D.)--The Johns Hopkins University, 2002.
520 $a In diploid yeast cells, the MATα2 and MAT<bold>a</bold>1 homeodomain proteins bind DNA cooperatively to repress transcription of haploid-specific genes. In haploid α cells, MATα2 binds DNA cooperatively with MCM1 to repress <bold>a</bold>-specific gene (<italic>asg</italic>) transcription. Although the α2 protein binds DNA in a similar manner as other homeodomain proteins, a triple alanine mutant, in which the DNA major groove-contacting side chains are mutated to alanines, shows context-dependent DNA binding <italic> in vitro</italic> and <italic>in vivo</italic>. This triple mutant, called α2-3A, does not binds DNA detectably by itself or in complex with MCM1. Paradoxically, α2-3A still binds DNA in complex with <bold>a</bold>1 with unreduced affinity and specificity. To resolve this paradox, I determined the crystal structure of the <bold>a</bold>1/α2-3A heterodimer bound to DNA. The structure shows that the triple mutation causes a collapse of the α2-3A/DNA interface, giving rise to a re-organized set of contacts between α2-3A and DNA, that accounts for the ability of α2-3A and <bold>a</bold>1 to recognize the wild type DNA sequence. Isothermal titration calorimetry experiments revealed that a much more favorable entropic component stabilizes the <bold>a</bold>1/α2-3A/DNA complex, as compared with the α2-3A/DNA complex. These results suggest that either some of the reorganized α2-3A/DNA interactions are missing when α2-3A binds DNA alone, or that the <bold>a</bold>1/α2-3A/DNA complex is much more flexible than the wild type complex. The combined structural and thermodynamic study provides an explanation for how partner proteins can influence the sequence specificity of a DNA binding protein.
520 $a In part two, I present studies that address the structural basis for the reduced DNA-binding affinity and specificity of <bold>a</bold>1 in the absence of α2. MAT<bold>a</bold>1 is converted into strong DNA binder by the interaction with the C-terminal tail of MATα2. To understand why MAT<bold>a</bold>1 binds DNA weakly, and how the MATα2 tail affects the affinity of MAT<bold>a</bold>1 for DNA, we determined the crystal structure of a maltose-binding protein (MBP)-<bold>a</bold>1 chimera that has DNA binding behavior similar to MAT<bold>a</bold>1. The overall MAT<bold>a</bold>1 conformation in the MBP-<bold>a</bold>1 structure, which was determined in the absence of α2 and DNA, is similar to that in the <bold>a</bold>1/α2/DNA structure. The sole difference is in the C-terminal portion of the DNA recognition helix of <bold>a</bold>1, which is flexible in the present structure. However, these residues are not in a location likely to be affected by binding of the α2 tail. The results argue against conformational changes in <bold>a</bold>1 induced by the tail of α2, suggesting instead that the α2 tail energetically couples the DNA binding of α2 and <bold>a</bold>1.
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