Ubiquitin, the Proteasome, and Dynamics at the Protein/DNA Interface

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dc.contributor.advisor Kodadek , Thomas en
dc.creator Nalley , Kip A . en
dc.date.accessioned 2010 -07 -12T18 :11 :24Z en
dc.date.accessioned 2014 -02 -19T22 :01 :48Z
dc.date.available 2010 -07 -12T18 :11 :24Z en
dc.date.available 2014 -02 -19T22 :01 :48Z
dc.date.issued 2006 -05 -16 en
dc.identifier.other en
dc.identifier.uri http : / /hdl .handle .net /2152 .5 /474 en
dc.description.abstract Recently it has been discovered that a mutant species of Gal4 , that contains a three amino acid change in a surface loop of the DNA binding domain , does not occupy the GAL 1 /10 promoter under Gal4 inducing conditions as measured by Chromatin Immunoprecipitation (ChIP ) assays . However , this protein , Gap71 , occupies the promoter similarly to Gal4 under non -inducing (poised ) conditions . Additionally this protein was found to be poorly ubiquitylated in vitro under conditions where Gal4 is ubiquitylated . In order to determine the mechanisms involved in the protein destabilization I have examined the properties of the individual mutations that comprise Gap71 . These experiments have revealed that serine 22 is a site of phosphorylation of the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation , possibly acting as part of the kinase recognition site . Mutation of either residue blocks Gal4 DBD phosphorylation , its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo . These data represent the first report of an essential phosphorylation event for this paradigmatic transcription factor . In addition , experiments were done to directly measure the dynamics of the Gal4 /DNA complex . To measure the dynamics I have exploited the system developed by Dr . D . Picard and others using the Gal4 DNA binding domain fused to the estrogen receptor ligand binding domain . Each of these constructs has been shown to be inactive until the addition of estradiol , when they are released and bind the Gal4 UAS . These constructs allow me to temporally control the appearance of a large quantity protein that is able to compete with the endogenous Gal4 for the UAS sites in the genome . Under non -inducing conditions , the results are consistent with a rapidly exchanging complex . However , upon induction , the Gal4 -promoter complexes "lock in" and exhibit long half -lives of one hour or more . Furthermore , pharmacological inhibition of proteasome -mediated proteolysis had little or no effect of Gal4 -mediated gene expression . These studies show that proteasome -mediated turnover is not a general requirement for transactivator function and , when considered in the context of previous studies , that different transactivator -promoter complexes can have widely different lifetimes . en
dc.format.medium Electronic en
dc.format.mimetype application /pdf en
dc.language.iso en en
dc.subject Chromatin Immunoprecipitation en
dc.subject Transcription Factors en
dc.subject Ubiquitin en
dc.title Ubiquitin , the Proteasome , and Dynamics at the Protein /DNA Interface en
dc.type.genre dissertation en
dc.type.material Text en
thesis.degree.name Doctor of Philosophy en
thesis.degree.level Ph .D . en
thesis.degree.discipline Biological Chemistry en
thesis.degree.grantor Graduate School of Biomedical Sciences en
thesis.degree.department en
dc.format.digitalOrigin born digital en
thesis.date.available 2006 -05 -15 en

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Ubiquitin, the Proteasome, and Dynamics at the Protein/DNA Interface. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /474 .

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