Reference: Palangat M, et al. (2012) Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA polymerase II. Transcription 3(3):146-53

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Abstract


Single-molecule studies of RNA polymerase II (RNAP II) require high yields of transcription elongation complexes (TECs) with long DNA tethers upstream and downstream of the TEC. Here we report on a robust system to reconstitute both yeast and mammalian RNAP II with an efficiency of ~80% into TECs that elongate with an efficiency of ~90%, followed by rapid, high-efficiency tripartite ligation of long DNA fragments upstream and downstream of the reconstituted TECs. Single mammalian and yeast TECs reconstituted with this method have been successfully used in an optical-trapping transcription assay capable of applying forces that either assist or hinder transcript elongation.

Reference Type
Journal Article
Authors
Palangat M, Larson M, Hu X, Gnatt A, Block S, Landick R
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