Purification of synchronized Escherichia coli transcription elongation complexes by reversible immobilization on magnetic beads

• Main Authors: Kelly, S.L (Author), Strobel, E.J (Author), Szyjka, C.E (Author)
• Format: Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology Inc. 2022
• Subjects: Biomolecules; Electrophoretic mobility; Elongation; Elongation complex; Escherichia coli; Fundamental tools; In-vitro; Ligands; Magnetic beads; Nucleoside triphosphates; Purification; Reversible immobilization; Riboswitches; RNA; RNA polymerase; Synchronization; Vitro studies; Vitro transcription
• Online Access: View Fulltext in Publisher

 Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more nucleoside triphosphates from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 50 leader sequence, called capture sequence 3-structure cassette 1 (C3-SC1), to reversibly capture synchronized TECs on magnetic beads. We first show, using electrophoretic mobility shift and high-resolution in vitro transcription assays, that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating nucleoside triphosphates), and compatible with solid-phase transcription; the yield of this purification is 8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP (5-aminoimidazole-4-carboxamide riboside 50-triphosphate)-sensing and ppGpp (guanosine-30,50bisdiphosphate)-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription. © 2022 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.