The biological functions of DNA and RNA are dependent upon highly specific and complex interactions with proteins. Elucidation of the mechanisms by which proteins recognize and manipulate nucleic acids in such fundamental processes as transcription, translation, replication, and recombination remains an area of intense research activity in structural biology. Despite recent progress in this area, the enormous diversity of protein- nucleic acid interactions means that many questions of fundamental important remain to be answered. Multi-dimensional NMR methods will be used to investigate the mechanisms of site-specific nucleic acid recognition by two key eukaryotic transcription factors that interact with DNA through different binding motifs, the zinc finger and the HMG domain. The proposed NMR structural studies of functional multi-finger subdomains of transcription factor IIIA, which are involved in both DNA and 5S RNA recognition, will provide new insights into the fundamental principles and diversity of nucleic acid recognition by zinc finger proteins in general and by TFIIIA in particular. The structure, dynamic and hydration will be determined for the complex formed between a protein (termed zf1-3) containing the first three zinc fingers of TFIIIA, which constitute the minimal domain for specific and high-affinity binding to DNA, and a DNA duplex encompassing the cognate site of the internal control region of the 5S RNA gene. Investigations of the effects of key linker mutations on the structure, dynamics, and DNA binding affinity of zf1-3 will be investigated. NMR structural studies of a protein containing zinc fingers 4-7 of TFIIIA, which constitute the minimal domain for high-affinity 5S RNA binding, will be undertaken. HMG domains are found in an important family eukaryotic transcriptional regulators that recognize DNA through interactions in the minor groove, accompanied by dramatic (up to 130 degrees) bending of the DNA duplex. The solution structure, dynamics, and hydration will be determined for the HMG domain of the lymphoid enhancer- binding factor LEF-1, both free and complexed to its specific DNA recognition site. These studies will not only reveal the structural basis for DNA recognition by LEF-1, but will provide valuable insights into HMG domain-DNA interactions in general as well as add significantly to our understanding of the minor-groove interactions that lead to DNA bending.
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