Experiments proposed here are designed to further our understanding of the structure and function of Transcription Factor IIIA (TFIIIA), with particular reference to its role in establishing sequence specificity for transcription initiation on 5S rRNA genes and its status as the prototype for the large class of nucleic acid-binding proteins containing Zn++ finger motifs. Specifically mutated forms of TFIIIA will be synthesized, purified, and characterized in a variety of in vitro assays to assess the importance of specific amino acid residues in contributing to structural stability in the Zn++ finger domains, and the role of different Zn++ fingers in mediating the interaction of TFIIIA with 5S rRNA and 5S rRNA genes, including 5S rRNA genes that are differentially regulated during development. The correlation between TFIIIA binding affinity and its ability to support transcription of 5S rRNA genes will also be analyzed. Experiments are proposed that may allow us to identify amino acid residues in TFIIIA that make direct contacts with the DNA helix, and a potential genetic approach for identifying TFIIIA mutants with altered DNA-binding affinity and/or specificity will be pursued. Finally, the ability to readily produce large amounts of very pure, active TFIIIA will be exploited in biophysical studies of TFIIIA structure and function in collaboration with other laboratories. Because of the particular relevance of TFIIIA to understanding 5S rRNA gene expression, and the more general importance of understanding the structure and function of Zn++ finger proteins, of which TFIIIA is the prototypical example, the proposed work can be expected to be of interest to a broad audience.
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