The objectives are to elucidate the structural and functional roles of Zn(II) in 1) zinc metalloenzymes, 2) the eukaryotic transcription factors originally termed zinc fingers, and 3) several proteins involved in nucleic acid replication which contain zinc. The specific protein molecules under investigation are: 1) alkaline phosphatase; 2) the fungal transcription factors GAL4, LAC9, MAL63, LEU3 and HAP1; 3) the glucocorticoid and estrogen receptors from the hormone receptor class of zinc transcription factors; and 4) the Zn-containing single stranded nucleic acid binding proteins including gene 32 protein from T4 and the retroviral nucleocapsid proteins binding to RNA. Three classes of zinc complex (zinc finger, zinc twist and zinc cluster) found in Zn-containing transcription factors are defined based on structural data available on this broad class of proteins. Cloning techniques are used to isolate the Zn-containing DNA binding subdomains of these large proteins (700-800 AA) as folded polypeptides of under 100 residues. Hence the powerful methods of 2D 1H NMR (COSY & NOESY) can be used for the determination of the solution structures of these domains to which the large transactivating domains are attached. Cd(II) is as effective as Zn(II) in inducing the specific conformations of these proteins required to recognize the specific DNA sequences. Substitution of 113Cd provides for the application of a series of heteronuclear NMR techniques which are valuable for determining the structures of the metal complexes. Zn-binding motifs have emerged as a major feature of the architecture of the subdomains which confer upon transcription factors the capacity to recognize specific DNA sequences. """"""""Zinc finger"""""""" proteins are essential in the process of development and differentiation (several Drosophila proteins controlling developmental destiny are zinc finger proteins), metabolic control (fungal transcription factors), and even oncogenesis (the erbA oncogene is an analogue of the thyroid hormone receptor). Understanding the structure of the DNA binding domains of these proteins and how the resultant interaction changes the structure of the DNA and places the attached trans activating domain into position in the eukaryotic transcription complex will be essential to our attempts to control or alter any of these processes when they become abnormal. There are now approximately 300 zinc enzymes characterized. They range from simple hydrolytic enzymes to RNA polymerases and are necessary for all aspects of metabolism.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Laughlin, Maren R
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Yale University
Schools of Medicine
New Haven
United States
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