Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24. Samples of uniformly "N-labeled and 15N,'3C-labeled recombinant proteins were used in the investigation. A near-complete assignment of the backbone 'H, '?N, and "C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional '5N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments. 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-I5N-edited and 'T-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations. The resulting three-dimensional structure contains a threehelix bundle and a small four-stranded antiparallel P-sheet that forms a hydrophobic core. The two Cterminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding. In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between p3 and p4. Thir leads to a tight turn between these p-strands.Keywords: NMR ; protein solution structure; DNA binding; heat-stress trancnption factor A central element of the rapid reprogramming of gene expression during the heat-stress response in all organisms is the activation of heat-stress genes. Newly formed heat-stress proteins are essential for the maintenance of the structural and functional integrity of cells during the stress period (Morimoto, 1993 ;Nover, 1991). With very few exceptions, heat-stress genes are characterized by a conserved palindromic recognition site in their promoter regions (5'-AGAANNTTCT-3'). The search for the corresponding transcription-activating proteins (heat-stress transcription factors, HSF) led to the identification of a new family of regulatory proteins with a N-terminal DNA-binding domain of about 100 amino acid residues which is conserved in yeast, insects, mammals and plants. (Scharf et al., 1990(Scharf et al., , 1994.Recently the three-dimensional structure of recombinant protein fragments of the yeast and Drosophila HSF has been investigated by means of X-ray crystallography (Harrison et al., 1994) and NMR techniques (Damberger et al., 1994; Vuister et al., 1994a,b). The DNA-binding domain contains a central helix-turn-helix motif which is thought to be responsible for specific DNA recognition (Vuister et al., 1994a Ahhrevinrions. DQF-COSY, double-quantum filtered correlation spectroscopy ; HMQC, heteronuclear-multiple-quantum-correlation spectroscopy ; HSE, heat-stress element; HSF, heat-stress transcription factor; HSQC, heteronuclear-single-quantum-correlation spectroscopy; TPPI, time-proportional phase incrementation.Enzyme. Thrombin (EC 3.4.21.5).remarkable similarities of all HSF, the three tomato proteins sequenced (Scharf et al...
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