Nucleic acid binding proteins are of major importance in biology since they include many of the factors that regulate gene expression and developmental processes. One major class of such factors are constituted by the family of zinc finger proteins, defined by conserved structural motifs which require zinc ions for appropriate folding into active forms. This project is focused on a genetic and molecular analysis of zinc finger proteins and their genes in different biological systems. (1) How many zinc finger proteins are encoded in the genome and how are these genes distributed on the chromosomes? This question is being asked in the human with regard to the C2H2 family of zinc finger proteins. Over 200 genomic clones have been isolated and are being sorted into groups of overlapping sequences that represent distinct loci, providing evidence for at least 20 finger loci in the human genome. Most of these loci have been mapped onto the human chromosome complement by in situ hybridization. By extrapolation to additional clones obtained in this and in other laboratories it can be estimated that the human genome contains at least one hundred, and possibly several hundred, genes for zinc finger proteins of the C2H2 class. (2) What is the structure and mechanism of interaction of the C2H2 finger domains with nucleic acids? This question is being addressed through a detailed structural analysis of the finger regions from several known nucleic acid binding proteins. A database of Zinc Finger proteins has been established for the purpose of statistical and structural modeling of the individual finger regions. Using constrained molecular dynamics (MD) to analyze the structural potentials for the finger domains from Xenopus laevis TFIIIa, mouse mkr2, and yeast Adr1, we have identified two general structural motifs. Fingers with primary structures that fit the consensus homology may fold to form short stable alpha to 3,10 helical regions on the histidine side of the domain, while nonconsensus fingers appear to have a more extended beta loop structure. Potential energy analysis from the MD simulations suggested that fingers 1 and 3 have the least and most stable structures, respectively, among TFIIIA finger domains. This structural instability has been confirmed through 1D 1H NMR spectroscopy of chemically synthesized TFIIIA finger 1.
