Extensive mutational analysis has revealed that a basal level of transcription is usually observed when all but a small region of DNA has been deleted from a eukaryotic gene promoter. These promoter elements that are necessary and sufficient for specific transcription initiation are referred to as minimal or core promoter elements. One common core element is the TATA box, located at about 30 nucleotides upstream from the initiation site of transcription. A less well-characterized, but perhaps equally important, initiator (Inr) constitutes a second such element that appears to be present in many RNA polymerase II transcribed genes. Although it has been demonstrated that, for many promoters, the integrity of the TATA element is essential for accurate and efficient transcription initiation, little is known about the role of Inr elements. The current proposed study focuses on the Inr element responsible for basal transcription of the human metallothionein IIA (hMTIIA) gene. Metallothionein are small, cysteine-rich proteins that are important in heavy metal detoxification and homeostasis. As appropriate for proteins that occupy such an essential physiological role, their expression is highly regulated. This is achieved by the presence of multiple, distinct metallothionein genes, each of which is regulated primarily at the transcriptional level. The sequence surrounding the initiation site of the hMTIIA gene exhibits strong homology to some of the previously identified Inr elements. Although expression of the hMTIIA gene is inducible by various agents, hMTIIA messenger RNAs are synthesized constitutively at high levels in most cells; therefore, its promoter provides an attractive model to study basal transcription regulation. The overall hypothesis to be evaluated is that the hMTIIA initiation sequence may be an important transcription regulatory element. The long- term goal of this project is to understand the hMTIIA Inr element, with particular emphasis on identifying cellular DNA-binding proteins that interact selectively with this Inr to regulate transcription. These studies will be of value not only in understanding the fundamentals of eukaryotic transcription regulation, but also will provide additional insights regarding the regulation of a physiologically important gene. With recombinant DNA techniques and standard promoter strength assay systems, the hMTIIA In element will be analyzed, both in its native promoter environment and after systematically modifying or linking it to heterologous promoters. The hypothesis that trans-acting factors may interact with the cis-acting hMTIIA Inr element to regulate transcription will be tested with protein-DNA binding assays. Knowledge gained from these experiments will be used to purify regulatory proteins that interact with the hMTIIA Inr and to clone the corresponding genes. Finally, the purified and clone proteins will be tested for their abilities to affect transcription initiation.
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