Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.

Observation of coordinated cotranscriptional RNA folding events.

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from RNA polymerase. Here, we have developed a concise, high-resolution cotranscriptional RNA chemical probing procedure called Transcription Elongation Complex RNA structure probing-Multilength (TECprobe-ML). We validated TECprobe-ML by replicating and extending previous analyses of ZTP and fluoride riboswitch folding, and mapped the folding pathway of a ppGpp-sensing riboswitch. In each system, TECprobe-ML identified coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-ML as an accessible method for mapping cotranscriptional RNA folding pathways.

Isolation of synchronized E. coli elongation complexes for solid-phase and solution-based in vitro transcription assays.

Synchronized transcription elongation complexes (TECs) are a fundamental tool for investigating the biochemical properties of RNA polymerases (RNAPs) and nascent RNA. We recently developed a standardized system for isolating high-purity synchronized E. coli RNAP TECs from an in vitro transcription reaction. Our system uses a custom 5' leader sequence, called C3-SC1 to immobilize synchronized TECs on magnetic beads so that free DNA and non-productive transcription complexes can be depleted. The synchronized elongation complexes isolated by our procedure, called C3-SC1TECs, are >98% active, >95% pure, and can be used in both solid-phase and solution-based transcription assays. The yield of the procedure relative to input DNA is ~11% when C3-SC1TECs are isolated for solid-phase assays and ~8% when C3-SC1TECs are isolated for solution-based assays. Here we describe protocols for purifying C3-SC1TECs, and for assessing the activity, homogeneity, and yield of C3-SC1TEC preparations.

Cotranscriptional RNA Chemical Probing.

Cotranscriptional folding is a fundamental step in RNA biogenesis and the basis for many RNA-mediated gene regulation systems. Understanding how RNA folds as it is synthesized requires experimental methods that can systematically identify intermediate RNA structures that form during transcription. Cotranscriptional RNA chemical probing experiments achieve this by applying high-throughput RNA structure probing to an in vitro transcribed array of cotranscriptionally folded intermediate transcripts. In this chapter, we present guidelines and procedures for integrating single-round in vitro transcription using E. coli RNA polymerase with high-throughput RNA chemical probing workflows. We provide an overview of key concepts including DNA template design, transcription roadblocking strategies, single-round in vitro transcription with E. coli RNA polymerase, and RNA chemical probing and describe procedures for DNA template preparation, cotranscriptional RNA chemical probing, RNA purification, and 3' adapter ligation. The end result of these procedures is a purified RNA library that can be prepared for Illumina sequencing using established high-throughput RNA structure probing library construction strategies.

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

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 5' 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 5'-triphosphate)-sensing and ppGpp (guanosine-3',5'-bisdiphosphate)-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.