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Transcription. 2012 May-Jun;3(3):146-53. doi: 10.4161/trns.20269.

Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA polymerase II.

Author information

1
Laboratory 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.

PMID:
22771949
PMCID:
PMC3616084
DOI:
10.4161/trns.20269
[Indexed for MEDLINE]
Free PMC Article
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