Localization of genes along the chromosomal DNA structure represents a crucial goal in the construction of genetic and physical maps and provides focal points for sequencing analysis of the human genome. The objective of this proposal is to develop methodology for producing and labeling proteins that recognize specific sequences in DNA that are associated with the initiation of gene transcription. Many transcription factors have been identified, including the TATA-binding protein (TBP) that is ubiquitous in eukaryotes. The TATA regulatory elements, which are recognized by TBP, are located upstream of the vast majority of functional genes distributed throughout the chromosomes. TBP labeled with a fluorescent tag (e.g., coumarin) will be expressed in a cell-free protein biosynthetic system. Coumarin-tagged TBP will then be tested for its ability to recognize TATA sites in cosmids, YACs and chromosomes. Fluorescence methods will be applied to measure the number and location of TATA sites in cloned fragments of DNA. In addition, efforts will be directed toward developing a useful DNA- cleaving agent by the attachment of such groups as EDTA Fe++ to the N- terminus of TBP. This would lead to the cleavage of DNA at all TBP-tagged sites. Such fragments offer special opportunities for cloning and sequencing functional genes.
