Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster.
TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.
Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
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CH
Wu
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Madabusi
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Nishioka
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Emanuel
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Sypes
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Arkhipova
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Gilmour
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D035362:Transcription Factor TFIID; D019473:Transcription Factors, TFII / Q000378:metabolism; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D035362:Transcription Factor TFIID; D019473:Transcription Factors, TFII; D004247:DNA; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase