TFIIIA binds with high affinity to the 5SrRNA gene as a specific transcription factor and to 5S RNA in a 7S RNP storage particle. This proposal focuses on both the DNA and RNA sequence elements required for high affinity interactions with TFIIIA and the roles played by individual Zn finger domains in specifying high affinity DNA and RNA binding. Different sets of Zn fingers are required for high affinity DNA and RNA binding. Experiments are proposed to investigate the structural elements in DNA and RNA recognized by TFIIIA as well as further investigation into the role of specific amino acids (within specific fingers and in the linker region between fingers). Mutagenesis phage display will be used to investigate the amino acid differences that distinguish RNA and DNA specific Zn fingers. Filter binding and fluorescent anisotrophy solution methods will be used to determine the energetic processes that drive binding reactions to DNA and RNA. Continued collaborative efforts will attempt to obtain high resolution structures of Zn finger DNA and RNA complexes by heteronuclear NMR methods and X-ray crystallography.
