Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase
Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase
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DOI：
10.1016/s0006-3495(99)77067-0
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发表时间：
1999-10-01
影响因子：
3.4
通讯作者：
Bustamante, C
中科院分区：
文献类型：
--
作者：
Guthold, M;Zhu, XS;Bustamante, C
用扫描力显微镜直接观察了非特异性和特异性的大肠杆菌RNA聚合酶(RNAP)-DNA复合体的动力学。为此，必须找到成像条件，在这种条件下，DNA分子吸附在云母上的强度足够强，可以成像，但又足够松散，能够扩散到云母表面。在非特异性复合体的连续图像中，可以看到RNAP沿着DNA滑动，执行一维随机行走。肝素是一种已知可以破坏非特异性RNAP-DNA相互作用的物质，它阻止了滑动。这些观察表明，RNAP沿DNA的扩散构成了一种加速启动子定位的机制。还研究了单个转录的RNAP分子的序列图像。当在液室中向停滞的延伸复合体中加入5MU的核苷三磷酸时，可以看到RNAP分子以1.5核苷酸/S的速率在与启动子方向一致的方向上穿梭其模板。用放射性标记的云母结合的转录复合体进行转录测试，证实了这个速率，大约比溶液中的复合体的速率小三倍。本实验还表明，停顿位点和终止位点的模式受到表面的影响。利用爱因斯坦-萨瑟兰摩擦扩散关系，估算和讨论了RNAP在DNA表面摩擦时所受的载荷力。
The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse an the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 mu M nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.