Schulz A, Mücke N, Langowski J, Rippe K
Abteilung Biophysik der Makromoleküle, Im Neuenheimer Feld 280, Heidelberg, D-69120, Germany.
J Mol Biol. 1998 Nov 6;283(4):821-36. doi: 10.1006/jmbi.1998.2131.
Scanning force microscopy (SFM) was used to visualize complexes of Escherichia coli RNA polymerase.sigma54 (RNAP.sigma54) and a 1036 base-pair linear DNA fragment containing the glnA promoter. In order to preserve the native hydration state of the protein-DNA complexes, the samples were injected directly into the SFM fluid cell and imaged in buffer. With this protocol, an apparent bending angle of 26(+/-34) degrees was determined for the specific complexes at the promoter. The bending angle of the unspecifically bound RNAP.sigma54 showed a somewhat broader distribution of 49(+/-48) degrees, indicating the existence of conformational differences as compared to the closed complex. In about two-thirds of the closed complexes, the RNA polymerase holoenzyme was located in a lateral position with respect to the DNA and the bend of the DNA was pointing away from the protein. This conformation was consistent with the finding that for the complexes at the promoter, the apparent contour length was reduced by only about 6 nm in buffer as compared to the free DNA. From these results we conclude that in the closed complex of RNAP. sigma54, the DNA was not wrapped around the polymerase, and we present a model for the trajectory of the DNA with respect to the RNA polymerase. The images acquired in buffer were compared to samples that were washed with water and then dried before imaging. Two artefacts of the washing and drying process were detected. First, extensive washing of the sample reduced the number of the specific complexes bound at the promoter (closed complex of RNAP.sigma54) from about 70% to 30%. This is likely to be a result of sliding of the RNAP.sigma54 holoenzyme along the DNA induced by the washing process. Second, the apparent DNA shortening of the contour length of RNAP.sigma54-DNA complexes at the promoter as compared to the contour length of the free DNA was 22 nm for the dried samples as opposed to only 6 nm for the undried samples imaged in buffer. This suggests an artefact of the drying process.
扫描力显微镜（SFM）用于观察大肠杆菌RNA聚合酶σ54（RNAP.σ54）与包含谷氨酰胺合成酶基因（glnA）启动子的1036个碱基对的线性DNA片段形成的复合物。为了保持蛋白质-DNA复合物的天然水化状态，将样品直接注入SFM流体池中并在缓冲液中成像。按照此方案，测定了启动子处特异性复合物的表观弯曲角度为26（±34）度。非特异性结合的RNAP.σ54的弯曲角度显示出更宽的分布，为49（±48）度，表明与封闭复合物相比存在构象差异。在大约三分之二的封闭复合物中，RNA聚合酶全酶相对于DNA位于侧向位置，并且DNA的弯曲方向远离蛋白质。这种构象与以下发现一致：对于启动子处的复合物，与游离DNA相比，缓冲液中的表观轮廓长度仅减少了约6nm。从这些结果我们得出结论，在RNAP.σ54的封闭复合物中，DNA没有缠绕在聚合酶周围，并且我们提出了一个关于DNA相对于RNA聚合酶轨迹的模型。将在缓冲液中获得的图像与用水洗涤然后在成像前干燥的样品进行比较。检测到洗涤和干燥过程的两个假象。首先，对样品进行大量洗涤后，启动子处结合的特异性复合物（RNAP.σ54的封闭复合物）数量从约70％减少到30％。这可能是洗涤过程诱导RNAP.σ54全酶沿DNA滑动的结果。其次，与缓冲液中成像的未干燥样品相比，干燥样品中启动子处RNAP.σ54-DNA复合物的轮廓长度表观缩短为22nm，而未干燥样品仅为6nm。这表明干燥过程存在假象。
