DNA Recognition by Basic-Leucine Zipper Proteins

EmBP-1是小麦碱性亮氨酸拉链（bZIP）类转录因子，参与基因表达的调控。拟议的实验将解决EmBP-1的DNA结合域的结构-功能关系。通过分析具有改变的结合偏好的突变DNA结合蛋白，可以了解bZIP蛋白DNA结合域内的氨基酸序列编码功能特异性的方式。EmBP-1二聚化和DNA结合功能的最小结构域将通过缺失分析和体外结合试验来确定。我们将构建编码随机突变基区结构域的cDNA文库，并用退化寡核苷酸进行探测，以确定bZIP结构域氨基酸序列的变化是如何在改变的结合特异性中表现出来的。这些发现的功能意义将通过在转基因拟南芥植物中表达诱变的EmBP-1来进行体内测试。此外，为了研究bZIP序列的自然变异，将从拟南芥中分离出EmBP-1基因家族的成员。由于亮氨酸拉链类转录因子从植物到动物都是保守的，因此从拟议的研究中获得的知识将适用于大多数真核细胞中运作的基本原理。EmBP-1是一种小麦DNA结合蛋白，参与控制基因表达。了解DNA结合蛋白识别特定DNA序列的方式将有助于更好地理解细胞激活基因表达的机制。这对于理解正常生长发育以及理解通常与人类疾病（如癌症）相关的异常基因表达具有重要意义。通过分析具有改变的结合偏好的突变DNA结合蛋白，可以了解这类蛋白的功能方式。随机诱变的Dna结合域将被构建和分析，以确定氨基酸序列的变化如何表现在改变的结合特异性中。这些发现将通过在基因工程植物中表达诱变的EnBP-1来验证。此外，为了检验DNA结合序列的自然变异，将从拟南芥（一种用于分子遗传学研究的模式植物物种）中分离相关基因。因为这种类型的蛋白质存在于从植物到动物的生物体中，所以从拟议的研究中获得的知识将适用于大多数细胞中运作的基本原理。

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.