MECHANISMS OF TRANSCRIPTIONAL REGULATION IN EUKARYOTES

University Of California San Diego, La Jolla CA

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Abstract

DESCRIPTION: The long-term objective of this project is to understand the mechanism of basal transcription by RNA polymerase II. This grant specifically proposes to use both biochemical and genetic approaches to study the function of a conserved, downstream basal promoter element termed the DPE (downstream promoter element). The DPE is a distinct 7 bp element that is located at about +30 (typically, from +25 to +34) relative to the transcription start site. It is present in many TATA-less promoters and is specifically bound by TFIID, but not by the TATA-binding protein (TBP). The available data suggest that the DPE is a conserved core promoter element that is, in many respects, a downstream counterpart to the TATA box. The specific aims are as follows. 1. Biochemical characterization of the DPE. These studies involved the analysis of the characteristics of functionally active DPE elements as well as the nature of the physical interaction of TFIID with TATA-less, DPE-containing promoters. 2. Purification and characterization of a DPE-specific basal transcription activity. Basal transcription of TATA-less, DPE-containing promoters requires an activity that is distinct from the factors that are sufficient for transcription of TATA-containing promoters (i.e., this activity is distinct from TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II). This aim is focused on the purification and characterization of this activity, which is tentatively named DSF (DPE-specific factor). 3. Analysis of the DPE in vivo in transgenic Drosophila. Enhancer trapping techniques will be used to gain insight into the biological role of the DPE. In particular, these experiments will test the ability of a broad range of enhancers to regulate transcription differentially from TATA-containing, DPE-less (TATA+ Inr+ DPE-) promoters relative to TAT-less, DPE-containing (TATA- Inr+ DPE+) promoters. The experiments proposed here would contribute to our fundamental understanding of gene expression and would be applicable to the study of human diseases, such as AIDS and many forms of cancer, that might be treated by the specific and directed control of transcription.