Functional Analysis of Transcriptional Activators in Living Cells

Project: Research project

Project Details

Description

Gilmour 9723537 A new approach for investigating the function of transcriptional activators in living cells will be developed. A collection of 6 factors and RNA polymerase II compose the general machinery that is required for initiation of transcription but the activity of the general machinery is regulated by DNA sequence-specific binding proteins called activators. Genomic fingerprinting provides a way to monitor protein-DNA interactions in living cells and recently a new method using permanganate has been developed in this laboratory to monitor the interactions of TFIID and RNA polymerase II on promoters that have been transformed into Drosophila. Permanganate footprinting of a transformed promoter can be done in intact salivary glands that have been dissected from larvae, providing a true picture of an in vivo situation. The specific aims of this project are: 1) to use available fly stocks to determine if a GAL4 based approach can be used to investigate how an activator affects the promoter interactions of TFIID and RNA polymerase II in living cells 2)to determine how Gal4-VP16 and Gal4(10147) affect target promoters carrying various numbers of Gal4-binding sites 3) determine what changes occur in promoter interactions when the activator is switched on or off by using derivatives of the Tet-repressor. New insights into determining mechanisms for the regulation of transcription in living cells will result from this work. Although tremendous progress has been made in the identification and biochemical characterization of factors involved in transcriptional regulation of gene expression, much of the work has been done using artificially contrived systems or by indirect analysis. The ability to analyze such interactions in living cells should be useful in obtaining a more complete understanding of how gene expression is regulated.

StatusFinished
Effective start/end date9/1/974/30/02

Funding

  • National Science Foundation: $345,000.00

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