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Molecular and Cellular Biology, March 2001, p. 1593-1602, Vol. 21, No. 5
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.5.1593-1602.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Analysis of Core Promoter Sequences Located Downstream from the TATA Element in the hsp70 Promoter from Drosophila melanogaster

Chwen-Huey Wu,1 Lakshmi Madabusi,2 Hokuto Nishioka,3 Peter Emanuel,4 Michael Sypes,1 Irina Arkhipova,5 and David S. Gilmour1,*

Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 168021; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 787422; National Institutes of Health, Bethesda, Maryland 20892-18303; U.S. Army ERDEC SCBRD-RT, Bioprocess Engineering Facility, Aberdeen Proving Ground, Aberdeen, Maryland 21010-54244; and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138-20925

Received 29 September 2000/Returned for modification 30 October 2000/Accepted 6 December 2000

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.

* Corresponding author. Mailing address: Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-8905. Fax: (814) 863-7024. E-mail: dsg11{at}psu.edu.

Molecular and Cellular Biology, March 2001, p. 1593-1602, Vol. 21, No. 5
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.5.1593-1602.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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