Abstract
A Drosophila heat-shock gene transcription factor (HSTF) has been shown to bind to three domains upstream from the TATA homology on a hsp 70 gene. The domain closest to the TATA homology consists of two contiguous binding sites with different binding affinities. Occupancy of the TATA homology proximal site (site 1) coordinates HSTF binding to the neighboring site (site 2) in a cooperative manner (Topol, J., Ruden, D. M., and Parker, C. S. (1985) Cell 42, 527-537). We have used alkylation interference and protection experiments to determine which residues within the binding sites are closely contacted by the HSTF. The contacts inferred from these studies included the residues present in the consensus sequence found in all HSTF binding sites and exhibit rotational symmetry, suggestive of a multimeric HSTF. By employing a gel electrophoresis separation technique we were able to resolve two protein-DNA complexes consisting of site 1 occupancy (complex A) and sites 1 and 2 occupancy (complex B). Analysis of these discrete species reveals that a subset of contacts within site 1 change upon HSTF binding to site 2, suggesting that a conformational change in the protein-DNA complex occurs. Implications for the activation of heat-shock gene transcription are discussed.
Highlights
(HSTF) has been shownto bind to three domains up- phila hsp 70 gene in vivo
The contacts inferred from these studies included the residues presentin the consensus sequence first occupied at low levels of Heat-shock transcriptionfactor gene-specific transcription factor (HSTF) followedby efficient HSTF binding to the critical second site
In this study we have investigated the spatial distribution of the HSTFon binding sites 1 and 2 by performing a series found inall HSTF binding sites and exhibit rotational of chemical DNA interference and protection experiments
Summary
From the Division of Chemistry and Chemical Engineering, CaliforniaInstitute of Technology, Pasadena, California 91125. Been determined that two HSTF binding sites are present, The domain closest to theTATA homology consists of both of which must be present for maximal transcription of twocontiguousbinding sites with differentbinding the hsp 70 genes in vitro. These two sites are occupied by a affinities. Cooperative bindspecies reveals that a subset of contacts within site 1 ing to site 2 results in aseries of interesting transitions inthe change upon HSTF binding tosite 2, suggesting that a contacts made by the HSTFwithin the binding domain These conformational change in the protein-DNA complex contact transitions suggest that conformational changes in occurs. The potential significance of these changes in the activation of the hsp 70 genes is discussed
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