Solution Structure of the DNA‐Binding Domain of the Tomato Heat‐Stress Transcription Factor HSF24

Abstract

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...

Identification and expression analysis of OsHsfs in rice

“…The DBD domain contained three α-helix bundles and a small four-stranded antiparallel β-sheet as previously described in LpHsf24 (Fig.1) (Schultheiss et al, 1996). There was a conserved intron located near the 3′-end of the third helix ( Fig.1) Further analyses of the HR-A/B domain revealed that there were, in the majority of cases, three or more repeated heptads in the HR-A domain and two incompletely repeated heptads in the HR-B domain (Fig.2).…”

An Autoregulatory Loop Controlling Arabidopsis HsfA2 Expression: Role of Heat Shock-Induced Alternative Splicing

“…Although the t-DBD of S-HsfA2 lacks strands b3 and b4, it contains the H2-T-H3 motif that is responsible for HSE recognition and binding based on analysis of the DBD structure of tomato (Solanum lycopersicum) HSF24 (Schultheiss et al, 1996). Thus, we hypothesized that S-HsfA2 might bind to the HSEs.…”

Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators

“…2) in the winged helix-turn-helix DNA binding domain (Harrison et al 1994;Vuister et al 1994;Schultheiss et al 1996), an adjacent 80 residue hydrophobic repeat (HR-A/B) essential for trimer formation (Sorger and Nelson 1989;Clos et al 1990;Peteranderl and Nelson 1992), and the carboxy-terminal transactivation domain (Chen et al 1993;Green et al 1995;Shi et al 1995;Zuo et al 1995;Wisniewski et al 1996). With the exception of the HSF in budding yeast and human HSF4, another hydrophobic repeat (HR-C) is located adjacent to the transactivation domain; this repeat has been suggested to suppress trimer formation by interacting with HR-A/B (Nakai and Morimoto 1993;Rabindran et al 1993).…”