TIME RESOLVED FLUORESCENCE ENERGY TRANSFER STUDIES OF TRANSCRIPTION COMPLEXES

我们已经开发出方法， 在多肽C-末端的荧光探针，在多肽 六聚组氨酸标签，在DNA和RNA中， 共振能量转移，以限定成对的 荧光探针定点导入转录 配合物 我们正在处理以下问题：（1）结构性问题 RNA聚合酶内亚基的组织（2）包裹程度 起始复合物中RNA聚合酶周围的DNA（3） 转录泡的模板和非模板链， 起始复合物（4）新生RNA在延伸中的路径 复合物（5）与调节因子的相互作用和（6）实时， 起始、延伸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.