TIME RESOLVED FLUORESCENCE ENERGY TRANSFER STUDIES OF TRANSCRIPTION COMPLEXES

我们已经开发了一些方法来专门介绍 多肽C末端的荧光探针，多肽的 在DNA和RNA中，我们使用的是荧光 定义成对之间距离的共振能量转移 定点定向导入转录的荧光探针 复合体。我们正在解决以下问题：(1)结构性问题 RNA聚合酶中亚基的组织(2)包裹的程度 DNA在RNA聚合酶周围的起始复合体(3)位置 转录泡的模板和非模板链 起始复合体(4)新生RNA的伸长途径 复合体(5)与调节因子的相互作用和(6)实时， 启动、延长和调节的单分子分析。

We have developed methods to site-specifically introduce fluorescent probes at polypeptide C-termini, at polypeptide hexahistidine tags, in DNA, and in RNA, and we are using fluorescence resonance energy transfer to define distances between pairs of fluorescent probes site-specifically introduced into transcription complexes. We are addressing the following issues (1) structural organization of subunits within RNA polymerase (2) extent of wrapping of DNA around RNA polymerase in initiation complexes (3) positions of the template and non-template strands of the transcription bubble in initiation complexes (4) path of the nascent RNA in elongation complexes (5) interactions with regulatory factors and (6) real-time, single-molecule analysis of initiation, elongation, and regulation.