Escherichia coli RNA polymerase activity observed using atomic force microscopy
Escherichia coli RNA polymerase activity observed using atomic force microscopy
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DOI：
10.1021/bi9624402
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发表时间：
1997-01-21
期刊：
影响因子：
2.9
通讯作者：
Hansma, PK
中科院分区：
文献类型：
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作者：
Kasas, S;Thomson, NH;Hansma, PK
采用流体轻敲模式原子力显微镜（AFM）观察了大肠杆菌RNA聚合酶（RNAP）转录两种不同线性双链DNA模板的过程。在连续AFM图像中，通过添加5'-三磷酸核糖核苷（NTPs），观察RNAP对DNA模板的易位来检测转录过程，将RNAP、dsDNA和新生RNA的三元复合物吸附在云母表面，在连续流动的缓冲液下成像。在引入所有四个ntp后，我们观察到一些DNA分子被拉过RNAP，一些与RNAP分离，而另一些则没有相对于RNAP移动。在NTP浓度下，转录速率约为0.5-2个碱基/秒，约为5 μ m。RNA转录物在液体中不明确成像。然而，在使用小单链（ss）环状DNA模板的实验中，一旦样品干燥并在空气中成像，就可以用拍打模式AFM观察到长达1或2微米的转录本，这证实了我们对RNAP吸附在云母上的转录活性的观察。这项工作表明，流体中敲打模式的发展使得使用AFM在分子水平上跟踪生物过程成为可能，并获得有关结合在表面上的单个分子活性变异性的新见解。
Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images, Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 mu M. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air, This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica, This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.