Abstract
A series of studies were performed to determine whether zinc finger peptides could efficiently repress transcription from RNA polymerase II promoters in vivo and to determine how such repression might depend on the position of the zinc finger binding site with respect to those of the TATA box or the initiator element. Promoter constructs were prepared with Zif268 binding sites inserted at various positions, and the activity of a reporter gene was measured in transfection studies. We found that the peptide containing the three zinc fingers of Zif268 could efficiently repress activated transcription when bound to a site near the TATA box (19-fold repression) or when bound to a site near the initiator element (18-fold repression). Repression was even more effective when the zinc finger peptide was bound to both of these sites (63-fold repression). Novel zinc finger peptides that had been selected via phage display also served as repressors of activated transcription, but repression with these proteins was somewhat less efficient than with the Zif268 peptide.
Highlights
A series of studies were performed to determine whether zinc finger peptides could efficiently repress transcription from RNA polymerase II promoters in vivo and to determine how such repression might depend on the position of the zinc finger binding site with respect to those of the TATA box or the initiator element
Transcriptional Repression by Zinc Finger Peptides; Effects of Binding Site Placement within the Promoter Region—We used transient cotransfection studies, with various promoter constructs coupled to a luciferase reporter, to test whether zinc finger peptides can repress transcriptional initiation and elongation from RNA polymerase II promoters in vivo
This paper is available on line at http://www.jbc.org from Zif268 was tested as a repressor, and the optimal binding site for this zinc finger peptide (5Ј-tGCGTGGGCGg-3Ј) was incorporated at various positions between the TATA box and the initiator element (Figs. 1 and 2A)
Summary
(Received for publication, July 24, 1997, and in revised form, September 3, 1997). From the Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. Novel zinc finger peptides that had been selected via phage display served as repressors of activated transcription, but repression with these proteins was somewhat less efficient than with the Zif268 peptide. Recent phage display experiments suggest that it may be possible to select zinc finger peptides that will recognize almost any desired target site on duplex DNA [8]. Despite this rapid progress in design, there has been relatively little information about how such novel proteins might be used to regulate transcription or about the differential effects of targeting the zinc finger peptides to various promoter regions. We show that novel zinc finger proteins selected via phage display can function effectively as repressors in vivo
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