NMR STRUCTURE OF ZINC FINGERS SPECIFIC FOR HIV RRE RNA

描述：RNA-蛋白质相互作用对基因至关重要 对导致人类疾病的病毒的监管。这样做的长期目标是 项目是了解新型锌相互作用的结构基础 手指蛋白与HIV-1 Rev反应元件(RRE)RNA的结合 关键调控蛋白Rev.的站点将利用这一知识 开发具有亲和力和亲和力的RRE结合蛋白 活细胞中的特异性。 使用强大的噬菌体展示方法，允许采样和评分 在大量蛋白质同时存在的情况下，锌指蛋白已经被 与HIV-1 RRE和茎环IIB特异结合的分离物。核磁共振结构 测定用于阐明锌指的结构和特性 RNA相互作用。增强和进一步了解RNA结合特性 对于锌手指，设计过程将使用结构信息来引导 体外选择以及传统的定点诱变实验。 锌指蛋白与RRE相互作用的分子细节 茎环IIB将通过核磁共振确定。 该提案的近期目标有三个方面。首先，要了解如何 这些工程蛋白通过识别结合来结合它们的RNA底物 负责RNA接触的位点和氨基酸。第二，提高他们的素质 底物亲和力，Germann博士使用核磁共振结构数据进行位点定向 诱变实验和结构导向的噬菌体展示筛选。最后， 锌指作为生物抑制剂的局限性和适用性 将通过选择锌指蛋白对抗新的 RNA靶标。设计的锌指的抑制活性将是 通过体外和体内试验测定。

DESCRIPTION: RNA-protein interactions are of paramount importance for gene regulation of viruses that cause human disease. The long term goal of this project is to understand the structural basis for the interaction of novel zinc finger proteins with the HIV-1 Rev responsive element (RRE) RNA, the binding site for the critical regulatory protein Rev. This knowledge will be exploited for developing small RRE binding proteins that possess both affinity and specificity in living cells. Using a powerful phage display approach that permits the sampling and scoring of a large number of proteins simultaneously, zinc finger proteins have been isolated which bind specifically to HIV-1 RRE and stem loop IIB. NMR structure determination is used to elucidate the zinc finger structure and specifics of RNA interaction. To enhance and further understand the RNA binding properties of zinc fingers, a design process will use structural information to guide in vitro selection as well as traditional site directed mutagenesis experiments. The molecular details of the interaction between zinc finger proteins and RRE stem loop IIB will be determined by NMR. The immediate goals of the proposal are three fold. First, to understand how these engineered proteins bind their RNA substrates by identifying binding sites and the amino acids responsible for RNA contact. Second, to improve their substrate affinity, Dr. Germann uses NMR structural data for site directed mutagenesis experiments and structure guided phage display selection. Finally, the limits and applicability of zinc fingers as inhibitors of biological processes will be determined by selection of zinc finger proteins against new RNA targets. The inhibitory activity of designed zinc fingers will be determined through in vitro and in vivo assays.