STRUCTURE/FUNCTION OF HIV TAT-TAR COMPLEX

PI计划使用TAT-TAR系统来开发技术 用电子探针研究蛋白质-RNA复合体的三维结构 各种新的物理和化学方法。他已经发展出 在化学上将螯合剂与独特的核苷酸连接的技术 合成的RNA，允许通过以下方法检测附近的残留物 金属络合剂介导的切割位点的鉴定 共轭。这项技术还将用于监测构象 当蛋白质因子结合时发生的变化。特定残留物 结合蛋白中的接触将由核糖核酸链确定。 作为蛋白酶的芹菜，通过光交联和 通过使用螯合氨基酸类似物插入到特定的 焦油结合的TAT多肽的位置。在后一种方法中，联系人 将通过克隆体介导的结合RNA的切割来确定。这个 PI还将结合到特定RNA位点的螯合剂转化为 用于转移到TAT上的受体的荧光能量供体 多肽。由这些方法确定的距离将用于 对可能的RNA-蛋白质结构设置约束。

The PI plans to use the TAT-TAR system for development of techniques to probe the 3-dimensional structure of protein-RNA complexes using a variety of novel physical and chemical approaches. He has developed techniques to link chelators to unique nucleotides in chemically synthesized RNA that permit determination of nearby residues by indentification of the sites of cleavage mediated by the metal-chelator conjugate. This technology will also be used to monitor conformational changes that occur upon binding of protein factors. Specific residue contacts in bound proteins iwll be determined by ribonucleotide-linked celators that serve as proteases, through photo-crosslinking and through the use of chelating amino acid analogs inserted into specific sites of TAR binding TAT peptides. In this latter approach contacts will be determined by clelate-mediated cleavage of the bound RNA. The PI will also convert chelators bound to specific RNA sites to fluorescence energy donors for transfer to acceptors attached to Tat peptides. The distances determined by these methods will be used to set constraints on possible RNA-protein structures.