PHOTOCHEMICAL & SPECTROSCOPIC PROBES OF RNA POLYMERASE

拟议的工作是绘制新生的领先端的路径 细菌酶表面的核糖核酸 聚合酶和核糖体。原位制备标记的RNA 叠氮光亲和标记，以及随后的光解和分析 标记的产品，将显示酶的哪些亚基与 不断增长的核苷酸链以及RNA的链长 与DNA模板分离。附着在RNA上的光谱探针和 其他网站将提供有关两地之间距离的补充信息 大分子复合体上的位置，以及对自由的限制 新生核糖核酸的运动。双官能团螯合剂，类似物 乙二胺四乙酸，可共价连接到 生物分子，将被用作多用途光谱探针。 特别是双功能络合剂中的发光稀土离子 附着在新生RNA上的核糖核酸将作为能量转移的供体 学习。此外，从络合的扩散增强的能量转移 在溶液中的稀土元素将揭示出前端的可及性 大分子中的新生RNA和发色探针分子 复合体。这些实验将提供有关分子的新知识。 DNA转录和转录-翻译耦合机制； 更好地理解这些基本的生命过程最终可能会受益 人类健康。

The proposed work is to map the path of the leading end of nascent ribonucleic acid (RNA) across the surface of the bacterial enzyme RNA polymerase and on to the ribosome. In situ preparation of RNA tagged with azide photoaffinity labels, and subsequent photolysis and analysis of labeled products, will show which subunits of the enzyme are contacted by the growing nucleotide chain and also the RNA chain length at which RNA separates from DNA template. Spectroscopic probes attached to the RNA and other sites will provide complementary information about distances between sites on the macromolecular complex, and restrictions on the freedom of movement of nascent RNA. Bifunctional chelating agents, analogs of ethylenediaminetetraacetic acid which can be covalently attached to biological molecules, will be used as multipurpose spectroscopic probes. In particular, luminescent lanthanide ions in bifunctional chelating agents attached to nascent RNA will be used as donors for energy-transfer studies. Also, diffusion-enhanced energy transfer from chelated lanthanides in solution will reveal the accessibility of the leading end of nascent RNA, and of chromophoric probe molecules, in macromolecular complexes. These experiments will provide new knowledge of the molecular mechanisms of DNA transcription and transcription-translation coupling; better understanding of these basic life processes may ultimately benefit human health.