Promoter function and in situ protein/DNA interactions upstream of the yeast HSP90 heat shock genes.
We have mapped in vivo protein/DNA interactions within the upstream regulatory regions of the two yeast HSP90 genes, and have begun mutagenizing footprinted sequences in an effort to identify the cis-acting determinants of heat shock transcription. Genomic footprinting of the HSP82 promotor using chemical and enzymatic nucleases reveals that irrespective of transcriptional state, the most proximal of three heat shock elements, HSE1, is occupied along both sugar-phosphate backbones as well as within its major groove, while the TATA box is bound along both sugar-phosphate backbones. Distorted DNA structure is associated with each constitutively bound factor: protein binding to HSE1 appears to induce a local A-form-like helical conformation, whereas occupancy of the TATA box is associated with strand-specific nuclease hypersensitivity of an adjacent polypurine tract. In situ mutagenesis experiments indicate that HSE1 is absolutely required for both basal and induced expression, and that basal transcription can be preferentially abolished by point mutations within this sequence. In contrast, point mutations within the TATA element have the reverse effect, as induced transcription is more significantly affected. Similar to HSE1 point mutants, we have found that basal transcription is preferentially repressed by an HMRE silencer element when it is transplaced approximately 1 kb upstream of the HSP82 start site. Finally, a complementary footprinting analysis of the upstream region of the constitutively expressed HSC82 gene reveals the presence of three discrete protein complexes. These map to the TATA box, the promotor-distal heat shock element, C.HSE1, and a novel sequence upstream of C. HSE1, suggesting that the 10-fold higher basal transcription of HSC82 stems, at least in part, from a non-HSE-binding factor.
Gross
D S
DS
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130.
Adams
C C
CC
English
K E
KE
Collins
K W
KW
Lee
S
S
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016385:TATA Box; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors