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dc.contributor.authorNelson, Michael Robert
dc.creatorNelson, Michael Roberten_US
dc.date.accessioned2013-03-08T22:08:43Z
dc.date.available2013-03-08T22:08:43Z
dc.date.issued2012
dc.identifier.urihttp://hdl.handle.net/10150/271634
dc.description.abstractThe Cro family of bacteriophage DNA-binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are -helical and mixed -helical/- sheet, respectively, despite sharing 40% sequence identity. Both proteins bind DNA using a helix-turn-helix (HTH) motif, however, the natural consensus DNA sequences of the proteins are different at three positions in each seven base-pair half site. Fluorescence anisotropy measurements showed that wild-type Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (K(d)) of 230 nM and 56 nM, respectively. Wild-type Pfl 6 bound its noncognate site with K(d) = 1.99 μM and wild-type Xfaso 1 did not bind its noncognate site. We introduced mutations into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. By exchanging the HTH sequence of the two proteins the specificity of binding was switched from cognate to noncognate consensus site. We found that the local sequence is the primary determinant in the DNA binding specificity for Xfaso 1 and Pfl 6, and the global conformation is not the major difference in binding specificity.
dc.language.isoenen_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.titleThe Role of Protein Sequence and Global Conformation in DNA Binding Specificity of Members of the CRO Familyen_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.grantorUniversity of Arizonaen_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.nameB.S.en_US
refterms.dateFOA2018-08-16T21:01:37Z
html.description.abstractThe Cro family of bacteriophage DNA-binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are -helical and mixed -helical/- sheet, respectively, despite sharing 40% sequence identity. Both proteins bind DNA using a helix-turn-helix (HTH) motif, however, the natural consensus DNA sequences of the proteins are different at three positions in each seven base-pair half site. Fluorescence anisotropy measurements showed that wild-type Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (K(d)) of 230 nM and 56 nM, respectively. Wild-type Pfl 6 bound its noncognate site with K(d) = 1.99 μM and wild-type Xfaso 1 did not bind its noncognate site. We introduced mutations into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. By exchanging the HTH sequence of the two proteins the specificity of binding was switched from cognate to noncognate consensus site. We found that the local sequence is the primary determinant in the DNA binding specificity for Xfaso 1 and Pfl 6, and the global conformation is not the major difference in binding specificity.


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