Margeat Emmanuel, Kapanidis Achillefs N, Tinnefeld Philip, Wang You, Mukhopadhyay Jayanta, Ebright Richard H, Weiss Shimon
Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095, USA.
Biophys J. 2006 Feb 15;90(4):1419-31. doi: 10.1529/biophysj.105.069252. Epub 2005 Nov 18.
Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.
利用配备交替激光激发的全内反射荧光显微镜，我们能够在单个固定的转录复合物中检测到流产起始和启动子逃逸。我们的方法利用荧光共振能量转移来监测掺入RNA聚合酶（RNAP）中的荧光探针与掺入DNA中的荧光探针之间的距离。我们观察到，在流产起始时，RNAP前沿与RNAP下游DNA之间的距离会有微小但可重复且与流产产物长度相关的减小，而在启动子逃逸时，距离会大幅减小。对流产起始的群体分布和单分子时间轨迹的检查表明，在一致启动子处，在饱和核糖核苷三磷酸浓度下，流产产物的释放是限速步骤（即流产产物的合成和RNAP活性中心的向前易位很快，而流产产物的解离和RNAP活性中心的向后易位很慢）。使用这种新方法获得的结果证实并扩展了从扩散单分子获得的结果，并为在整个转录过程中实时、单分子观察转录复合物不同状态之间的转变铺平了道路。
