Fulcher, Timothy K.; (1996) The biophysical and biochemical characterisation of recombinant AP1 and a structural determination of two DNA binding sites, the TRE and CRE. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The experimental work of this thesis was carried out on the DNA binding domain of the Fos and Jun proteins to further characterise transcription factor function. The leucine coiled-coil and basic region (the DNA binding domain) were overexpressed and purified to homogeneity using metal chelation affinity. The DNA binding specificity was characterised by gel retardation studies. It was shown that the binding of the wbFos:wbJun heterodimer to an oligodeoxynucleotide containing the AP1 DNA binding site was specific. Reducing conditions (5 mM DTT) significantly improved the specific shift observed. The specific binding was demonstrated by competition assays using oligodeoxynucleotides containing the DNA binding sites of other transcription factors. 53 amino acid residues were helical in the wbFos:wbFos homodimer, 35 in the wbJun:wbJun homodimer and 36 amino acid residues in the wbFos:wbJun heterodimer. In a buffer containing 50 [percent] trifluoroethanol the helical content of the wbFos:wbFos homodimer increased by 20 amino acid residues, consistent with the folding of the basic region. The relative stability of the dimers, estimated from the van't Hoff enthalpy, showed that the heterodimer was significantly more stable than the wbFos:wbFos homodimer. The Gibb's free energy at 310 K for the wbFos:wbFos homodimer was 1.6 kJ/mol, with a measured Kd of 5-10 [mu]M. The wbJun:wbJun homodimer had a Gibb's free energy at 310 K of -5.3 kJ/mol and a about 0.75 [mu]M. The Gibb's free energy at 310 K for the the wbFos:wbJun heterodimer had an enthalpy of unfolding of -171 kJ/mol and a Kdmuch less than 0.10 [mu]M. This suggests that the preferential formation of the heterodimer was through the destabilisation of the wbFos:wbFos homodimer. Two oligodeoxynucleotides containing the AP1 and GCN4 DNA binding site were characterised by high resolution NMR studies. 114 out of 124 protons were assigned for the symmetric tetradecamer d(GCATGACGTCATGC)2 containing the consensus GCN4\DNA binding site. The pseudorotation phase angle of the nucleotides was 144[degrees] for pyrimidines and 162[degrees]for purines. The fraction in the south conformation of nucleotides G1 to G13 was greater than 80[percent]. The pseudorotation phase angle of the 3' terminal nucleotide (C14) was 54[degrees]. The conformation of the base relative to the nucleotide (X), was typical of B-DNA for all the nucleotides G1 through G13. The correlation time for the tetradecamer of 3.5 +/- 0.05 ns was measured from the cytosine (C7 and C10) H5-H6 NOE timecourse. Further characterisation of the symmetric tetradecamer compared the relaxation rate constants for number of the protons at different spectrometer frequencies. The intrinsic relaxation rate constants (p) showed little variation with spectrometer frequency, whilst the initial slopes determined from the inversion recovery timecourse significantly varied with spectrometer frequency. The initial slopes were also very different from the apparent R1 values determined from the inversion recovery timecourse. The recovery timecourses appeared monoexponential. Adenine H2 showed a pronounced lag in the recovery. The effect of a short recycle time diminished the cross peak intensity. However the saturation factors were similar and normalised cross peaks showed little dependence on the recycle time. The protons of the pentadecamer containing the consensus AP1 DNA binding site, 5'd(CAAGTGACTCAGCGC):d(GCGCTGAGTCACTTG), were incompletely assigned. CD demonstrated that the overall conformation of both oligodeoxynucleotides was B- DNA.

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