Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization.

The ability to prepare defined transcription elongation complexes (TECs) is a fundamental tool for investigating the interplay between RNA polymerases (RNAPs) and nascent RNA. To facilitate the preparation of defined TECs that contain arbitrarily long and complex transcripts, we developed a procedure for isolating roadblocked E. coli TECs from an in vitro transcription reaction using solid-phase photoreversible immobilization. Our approach uses a modified DNA template that contains both a 5' photocleavable biotin tag and an internal biotin-TEG transcription stall site. Because the footprint of a TEC at the stall site sequesters the biotin-TEG tag, DNA template molecules that contain a TEC can be reversibly immobilized on streptavidin-coated magnetic beads by the 5' photocleavable biotin tag. In contrast, DNA template molecules that do not contain a TEC are retained on the beads because the biotin-TEG tag is exposed and can bind streptavidin. In this way, DNA template molecules that contain a TEC are gently separated from free DNA and DNA that contains non-productive transcription complexes. This procedure yields precisely positioned TECs that are >95% pure with >30% yield relative to the amount of input DNA template. The resulting complexes are >99% stable for at least 3 h and can be used for biochemical investigations of nascent RNA structure and function in the context of E. coli RNAP. The procedure is likely generalizable to any RNAP that arrests at and sequesters the internal biotin-TEG stall site.

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.

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.

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.

Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking.

Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription "roadblocking" approaches halt transcription elongation using a protein blockade that is non-covalently attached to the template DNA. I previously developed a strategy for halting Escherichia coli RNAP at a chemical lesion, which expands the repertoire of transcription roadblocking technologies and enables sophisticated manipulations of the arrested elongation complexes. To facilitate this chemical transcription roadblocking approach, I developed a sequence-independent method for preparing internally modified dsDNA using PCR and translesion synthesis. Here, I present a detailed protocol for the preparation and characterization of internally modified dsDNA templates for chemical transcription roadblocking experiments. Graphic abstract: Precise transcription roadblocking using functionalized DNA lesions.

Efficient Linear dsDNA Tagging Using Deoxyuridine Excision*.

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3' ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3' overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5' overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.

Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads.

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin-triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.

Chemical roadblocking of DNA transcription for nascent RNA display.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription "roadblocking" approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control.

Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways, and the mechanisms that enable transit between them, remain largely unknown. Here, we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA molecules navigate cotranscriptional folding pathways.

SnapShot: RNA Structure Probing Technologies.

Chemical probing coupled to high-throughput sequencing offers a flexible approach to uncover many aspects of RNA structure relevant to its cellular function. With a wide variety of chemical probes available that each report on different features of RNA molecules, a broad toolkit exists for investigating in vivo and in vitro RNA structure and interactions with other molecules.

High-throughput determination of RNA structures.

RNA performs and regulates a diverse range of cellular processes, with new functional roles being uncovered at a rapid pace. Interest is growing in how these functions are linked to RNA structures that form in the complex cellular environment. A growing suite of technologies that use advances in RNA structural probes, high-throughput sequencing and new computational approaches to interrogate RNA structure at unprecedented throughput are beginning to provide insights into RNA structures at new spatial, temporal and cellular scales.

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.

Cotranscriptional folding of a riboswitch at nucleotide resolution.

RNAs can begin to fold immediately as they emerge from RNA polymerase. During cotranscriptional folding, interactions between nascent RNAs and ligands are able to direct the formation of alternative RNA structures, a feature exploited by noncoding RNAs called riboswitches to make gene-regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function. To access cotranscriptional folding at nucleotide resolution, we extended selective 2'-hydroxyl acylation analyzed by primer-extension sequencing (SHAPE-seq) to measure structural information of nascent RNAs during transcription. Using cotranscriptional SHAPE-seq, we determined how the cotranscriptional folding pathway of the Bacillus cereus crcB fluoride riboswitch undergoes a ligand-dependent bifurcation that delays or promotes terminator formation via a series of coordinated structural transitions. Our results directly link cotranscriptional RNA folding to a genetic decision and establish a framework for cotranscriptional analysis of RNA structure at nucleotide resolution.

RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs.

RNAs assume sophisticated structures that are active in myriad cellular processes. In this review, we highlight newly identified ribozymes, riboswitches, and small RNAs, some of which control the function of cellular metabolic and gene expression networks. We then examine recent developments in genome-wide RNA structure probing technologies that are yielding new insights into the structural landscape of the transcriptome. Finally, we discuss how these RNA 'structomic' methods can address emerging questions in RNA systems biology, from the mechanisms behind long non-coding RNAs to new bases for human diseases.

A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing.

The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the 'elemental pause sequence' recognized from its frequent occurrence in the E. coli genome. σ70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause.

Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).

RNA molecules adopt a wide variety of structures that perform many cellular functions, including, among others, catalysis, small molecule sensing, and cellular defense. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally, we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

Two transcription pause elements underlie a σ70-dependent pause cycle.

The movement of RNA polymerase (RNAP) during transcription elongation is modulated by DNA-encoded elements that cause the elongation complex to pause. One of the best-characterized pause sequences is a binding site for the σ(70) initiation factor that induces pausing at a site near lambdoid phage late-gene promoters. An essential component of this σ(70)-dependent pause is the elemental pause site (EPS), a sequence that itself induces transcription pausing throughout the Escherichia coli genome and underlies other complex regulatory pause elements, such as the ops and his operon pauses. Here, we identify and provide a detailed kinetic analysis of a transcription cycle analogous to abortive cycling that underlies the σ(70)-dependent pause. We show that, in σ(70)-dependent pausing, the elemental pause acts primarily to modulate the rate at which complexes attempt to disengage the σ(70):DNA interaction. Our findings establish the σ(70)-dependent pause-encoding region as a multipartite element in which several pause-inducing components make distinct mechanistic contributions to the induction and maintenance of a regulatory transcription pause.