SPECIFICITY OF DNA BINDING BY ZINC FINGER PROTEIN

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Transcription factor IIIA (TFIIIA) is the prototype zinc finger nucleic acid-binding protein and is specifically required for transcription of the 5S RNA genes in the amphibian Xenopus. Although this protein has been studied extensively in terms of DNA binding and transcriptional activity, detailed structural information is still lacking on this important protein- DNA complex. Attempts at crystallization of the full length, nine zinc finger protein or the protein-DNA complex have not been successful. From derivatives of the cDNA clone for TFIIIA, we will produce, by overexpression in bacteria, truncated versions of TFIIIA protein. These polypeptides will be purified and tested for DNA binding activity. From these studies, we will determine the minimum number of zinc fingers required for sequence-specific binding. The thermodynamic properties of these complexes (association constants and half-lives) will be determined. We will use oligonucleotide-directed site-specific mutagenesis to alter specific amino acids in the critical zinc fingers of TFIIIA polypeptides and study the effects of these mutations on DNA binding. Mutations will be made in both finger and linker regions of the polypeptides. We will use short double stranded deoxyribo-oligonucleotides to define the minimal DNA sequences required for high affinity DNA sequence-specific binding. The effects of specific mutations on polypeptide binding will be assessed. TFIIIA polypeptide and mutant polypeptide complexes with these oligonucleotides will be prepared in sufficient quantity for two dimensional high resolution nuclear magnetic resonance spectroscopy. We will use UV-crosslinking with photoactive synthetic DNA probes to map the amino acid residues of TFIIIA involved in specific DNA interactions. These studies will extend our knowledge of the specific interaction of TFIIIA with the 5S RNA genes and provide insights into the general mechanisms of protein-DNA interactions.