About one in five genes of eukaryotic organisms lack a TATA box in the promoter to specify the start of transcription. We work on two genes with such TATA-less promoters, the human Ha-ras protooncogene and the proximal promoter of the major cytoplasmic actin gene of Drosophila, actin 5C. Our goal is to determine what selects the start sites in these promoters and to see how regulatory elements function in the activation of these TATA-less promoters. Though we will work on just two examples of TATA-less promoters, one human and one insect, our results should contribute to understanding the expression of TATA-less genes in general. The solution of specific health- related problems often depends on the results of such basic research in molecular biology. Start site selection in a TATA-less gene can be due either to a TATA- surrogate or it can be due to an initiator at the start site or, in some cases, to the two working together. The human Ha-ras proto-oncogene has multiple start sites of differing strengths mainly scattered over a 40 base pair region. We will make a variety of mutations of the start site region and upstream region of the Ha-ras gene abolishing elements or shifting their relative position. Then we will use primer extension to determine the start sites of transcripts formed from these mutant constructions transfected into cultured cells. A mutation moving a TATA-surrogate will move the start site the same amount. If a mutation of the start site itself eliminates expression, then an initiator is acting as selector. Thus we can determine whether an upstream selector or initiator, or both, selects each of the multiple Ha-ras start sites and identify the selecting element. We will try to see why just one of the multiple GC boxes, GCII at -150, functions as a major activating element of the Ha-ras promoter. We will try to locate a collaborating downstream element. In the Drosophila actin 5C proximal promoter we have already located an essential protein-binding element at -40 from the major start site. We will find out if this is a TATA-surrogate as above or just an activating element. If it is a TATA-surrogate, we will isolate the cDNA for binding protein, sequence this, and compare the encoded protein with the TATA- binding protein TFIID. Most Drosophila genes have a conserved sequence at the start site, but it is not known if this functions as an initiator. We will mutate this in the actin 5C proximal promoter and see the effect on start site selection.
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