title: Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA polymerase II.
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.
authors: Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD, USA. murali.palangat@nih.gov
authors: Murali
authors: M
authors: Palangat
authors: Matthew H
authors: MH
authors: Larson
authors: Xiaopeng
authors: X
authors: Hu
authors: Averell
authors: A
authors: Gnatt
authors: Steven M
authors: SM
authors: Block
authors: Robert
authors: R
authors: Landick
mesh_terms: D000818:Animals; D001483:Base Sequence; D053938:DNA Fragmentation; D008322:Mammals / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae