Observation by fluorescence microscopy of transcription on single combed DNA

被引:82
作者
Gueroui, Z [1 ]
Place, C [1 ]
Freyssingeas, E [1 ]
Berge, B [1 ]
机构
[1] Ecole Normale Super Lyon, Phys Lab, CNRS, UMR 5672, F-69364 Lyon, France
关键词
D O I
10.1073/pnas.092561399
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Molecular combing is a powerful procedure for aligning a large array of DNA molecules onto a surface. This technique usually leads to an overstretching of about 150% of the molecules' contour length. By changing the magnitude of capillary forces during the combing process, we were able to reduce the relative extension of the DNA molecules. Thus we achieved combing of T7 DNA with an extension close to its molecule contour length. We checked the ability of combed DNA to interact with DNA binding proteins. Using the T7 bacteriophage transcription system, we investigated the transcription activity of RNA polymerase on combed DNA by direct visualization of newly synthesized fluorescent RNAs. Our experiments show that no transcription activity occurs on overstretched DNA molecules, whereas we observe a transcription activity for nonoverstretched molecules. This activity is observed both in multiple initiation experiments and for one immobilized T7 RNA polymerase per promoter. These results open possibilities for the study of single enzyme actions on combed DNA by optical methods.
引用
收藏
页码:6005 / 6010
页数:6
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