DNA Recognition by Basic-Leucine Zipper Proteins

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EmBP-1 is a wheat basic-leucine zipper (bZIP) class transcription factor which has been implicated in the control of gene expression. Proposed experimentation will address the structure-function relationships of the DNA binding domain of EmBP-1. By analyzing mutant DNA binding proteins with altered binding preferences, insight can be gained of the ways in which amino acid sequences within the DNA binding domains of bZIP proteins encode functional specificities. The minimal domains for the dimerization and DNA binding functions of EmBP-1 will be determined using deletion analysis and in vitro binding assays. cDNA libraries encoding randomly mutagenized basic region domains will be constructed and probed with degenerate oligonucleotides to determine how changes in the amino acid sequences of the bZIP domains are manifested in altered binding specificities. The functional significance of these findings will be tested in vivo by expressing mutagenized versions of EmBP-1 in transgenic Arabidopsis plants. Additional, to examine the natural variation in bZIP sequences, members of the EmBP-1 gene family will be isolated from Arabidopsis. Because the leucine zipper class of transcription factors has been conserved from plants to animals, the knowledge gained from the proposed research will be applicable to the basic principles operating in most eukaryotic cells. %%% EmBP-1 is a wheat DNA binding protein involved in controlling gene expression. Understanding the way in which DNA binding proteins recognize specific DNA sequences will lead to a greater understanding of the mechanisms by which cells activate gene expression. This has great significance in understanding normal growth and development as well as in understanding aberrant gene expression which is often associated with human disease such as cancer. By analyzing mutant DNA binding proteins with altered binding preferences, insight can be gained of the ways in which this class of proteins function. Randomly mutagenizeds Dna binding domains will be constructed analyzed to determine how changes in the amino acid sequences are manifested in altered binding specificity. These findings will be tested by expressing mutagenized versions of EnBP-1 in genetically engineered plants, Additionally, to examine the natural variation in DNA binding sequences, related genes will be isolated from Arabidopsis (a model plant species used in molecular genetic studies). Because this type of protein is found within organisms ranging from plants to animals, the knowledge gained from the proposed research will be applicable to the basic principles operating in most cells.