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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Base Sequence
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DNA Fragmentation
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Mammals / genetics
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Mammals / metabolism
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Molecular Sequence Data
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Analysis, DNA
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Transcription, Genetic
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Transcriptional Elongation Factors / genetics
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Transcriptional Elongation Factors / metabolism*
Substances
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Saccharomyces cerevisiae Proteins
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Transcriptional Elongation Factors
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RNA Polymerase II
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RPB1 protein, S cerevisiae