Oligomerization properties of GCN4 leucine zipper mutants Db an in vivo study /
Leucine zippers (LZs) are dimerization motifs found in many
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1996.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739364591&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Leucine zippers (LZs) are dimerization motifs found in many eukaryotic transcription factors. The LZ consists of 4 to 6 heptad repeats, designated (abcdefg)nThey form parallel, a- helical dimers (coiled-coils). The d positions are usually occupied by Leu, while the a and d positions form a 4-3 hydrophobic repeat that is buried in the dimer interface. Many of the e and g positions are occupied by charged residues and also involved in intersubunit contacts. The LZ is not only of biological importance, but also provides a model system for structural studies. LZ fusions to the DNA- binding domain of k repressor were used to examine how mutations in the LZ from GCN4 affect its dimerization specificity. The mutants were characterized in terms of their oligomerization stabilities and specificities, using several in vivo assays developed on the basis of phage genetics. Expression level-sensitive repressor activity reflects the oligomerization stability. Cooperative DNA- binding to a pair of k operators distinguishes higher order oligomers from dimers. Specificity-dependent inhibition of repressor activity by coexpressed dominant negative variants detects heterodimer formation. Altering the position of a buried intersubunit Asn-Asn hydrogen bond within the a positions is sufficient to alter the dimerization specificity of the LZ. Certain changes at the e and g positions result in formation of higher order oligomers with strong cooperativity, and among them 3 consensus sequences were found. Dimeric e and g mutants have a variety of specificities, which do not correlate with the presence or number of putative intersubunit electrostatic interactions. The implications of these results for protein-protein interactions, de novo protein design, and protein folding in general are discussed. During the course of the studies, other experimental tools were also developed. These include: a shuttle system that allows construction of single-copy operon fusions, with both cat and lacz as reporters; 45 mutants of the lacUV5 promoter that drive different expression levels constitutively; and a Flag-M2 epitope tag engineered in the linker region of % repressor. These tools and the assays mentioned above win be useful for other studies of protein-protein and protein-DNA interactions. |
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| Item Description: | Vita. "Major Subject: Biochemistry". In title, numerals are used. |
| Physical Description: | xii, 127 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 92-107. |