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Updated: May 20, 2026

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Published on: May 13, 2019
Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA
Murali Palangat1, Matthew H Larson, Xiaopeng Hu
1Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD, USA. murali.palangat@nih.gov
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.
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