Systematic analysis of cotranscriptional RNA folding using transcription elongation complex display.

RNA can fold into structures that mediate diverse cellular functions. Understanding how RNA primary sequence directs the formation of functional structures requires methods that can comprehensively assess how changes in an RNA sequence affect its structure and function. Here we have developed a platform for performing high-throughput cotranscriptional RNA biochemical assays, called Transcription Elongation Complex display (TECdisplay). TECdisplay measures RNA function by fractionating a TEC library based on the activity of cotranscriptionally displayed nascent RNA. In this way, RNA function is measured as the distribution of template DNA molecules between fractions of the transcription reaction. This approach circumvents typical RNA sequencing library preparation steps that can cause technical bias. We used TECdisplay to characterize the transcription antitermination activity of 32,768 variants of the Clostridium beijerinckii pfl ZTP riboswitch designed to perturb steps within its cotranscriptional folding pathway. Our findings establish TECdisplay as an accessible platform for high-throughput RNA biochemical assays.

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.

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.