PHOTOCHEMICAL & SPECTROSCOPIC PROBES OF RNA POLYMERASE

长期目标是理解RNA作为其 合成是由DNA依赖的RNA聚合酶催化的，为了鉴定 信使RNA在执行其功能时遇到的细胞大分子 功能。具体目标是(1)扩展我们最近开发的 光亲和标记法测定所有大分子 当它通过真核生物时被不断增长的RNA链联系 转录复合体(HeLa RNA聚合酶II)；(2)研究 原核(大肠杆菌)转录本与核糖体等的相互作用 翻译设备的组成；(3)阐明位置 关于大肠杆菌RNA聚合酶亚基Sigma的初级序列 在转录复合体中被新生RNA的前端接触； 以及(4)根据需要设计和合成新的光亲和探针 目标1、2和3。这些探测器将具有不同的结构，它们将 不同的化学反应能力(氮化物、卡宾、硫氧尿嘧啶)，它们将 被合并到不同的核苷酸类似物中。它们的结合使用将 提供有价值的交叉核对和有关环境的新信息 RNA在其合成的每一步中。 基因表达的控制对健康和疾病都很重要，对两者都是如此 人类(真核)和细菌(原核)细胞。基因是 由依赖DNA的RNA聚合酶转录。计划中的实验将 为基因转录的分子机制提供新的知识， 转录-翻译耦合。更好地理解这些 基本的生命过程最终将造福人类健康。

The long-term objectives are to understand the path that RNA follows as its synthesis is catalyzed by DNA-dependent RNA polymerase, and to identify the cellular macromolecules that messenger RNA encounters in carrying out its functions. Specific aims are (1) to extend our recently developed photoaffinity labeling techniques to determine all the macromolecules contacted by a growing RNA chain as it passes through a eukaryotic transcription complex (HeLa RNA polymerase II); (2) to study the interactions of prokaryotic (E. coli) transcripts with ribosomes and other components of the translational apparatus; (3) to elucidate the positions on the primary sequence of the E. coli RNA polymerase subunit Sigma that are contacted by the leading end of nascent RNA in transcription complexes; and (4) to design and synthesize new photoaffinity probes as needed for aims 1, 2 and 3. These probes will have different structures, they will differ in chemical reactivity (nitrene, carbene, thiouracil), and they will be incorporated into different nucleotide analogs. Their combined use will provide valuable cross-checks and new information about the environment of RNA during each step of its synthesis. The control of gene expression is important in health and disease, in both human (eukaryotic) and bacterial (prokaryotic) cells. Genes are transcribed by DNA-dependent RNA polymerases. The planned experiments will provide new knowledge of the molecular mechanisms of gene transcription, and of transcription-translation coupling. Better understanding of these basic life processes will ultimately benefit human health.