Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597458
Title: Structural and functional studies of a transcription regulatory complex
Author: Chang, J.-F.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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Abstract:
The expression of immunoglobulin genes is controlled in part by the broadly expressed DNA-binding protein Oct-1 and the B cell-specific transcription co-activator, Bob1 (also known as OCA-B or OBF-1) that, together, form a complex on the immunoglobulin gene promoter. Bob1 interacts with the DNA-binding domain (the POU domain) of Oct-1 to specifically recognise the octamer DNA sequence, ATGCAAAT found in he promoter region. This thesis describes biophysical studies of the assembly of this complex. First, the binding of Oct-1 POU to the octamer element has been characterised using isothermal titration calorimetry (ITC). The results are correlated with the available structural information from the crystal structure of the Oct-1 POU/DNA complex. Formation of the Oct-1 POU/DNA complex is associated with a large negative heat capacity change, which suggests processes such as protein folding and surface dehydration are coupled to the protein-DNA interactions. The binding constant is observed to have a strong salt-dependence, and quantitative analysis suggests that there are six-ion pairs involved in the interaction, which is in good agreement with the crystallographic structural data. The enthalpy changes associated with ionisation indicate that carboxylate groups become protonated on the specific protein/DNA complex. We propose the Asp41, Glu51 and Glu143, which have been shown to be in the vicinity of the DNA phosphate backbone, are neutralised on complex formation. The second aim of the project was to analyse the structure of the POU/DNA/Bob1 ternary complex. Initially, an aim was to over-express Bob1 to crystallise the protein in the presence of the Oct-1 POU domain and DNA in order to study the protein-protein and protein-DNA interactions by X-ray crystallography.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.597458  DOI: Not available
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