RNA Folding During Transcription: Protocols and Studies

doi:10.1016/S0076-6879(09)68009-5

Methods in Enzymology

Volume 468, 2009, Pages 167-193

https://doi.org/10.1016/S0076-6879(09)68009-5 Get rights and content

Abstract

RNA folds during transcription in the cell. Compared to most in vitro studies where the focus is generally on Mg²⁺-initiated refolding of fully synthesized transcripts, cotranscriptional RNA folding studies better replicate how RNA folds in a cellular environment. Unique aspects of cotranscriptional folding include the 5′- to 3′-polarity of RNA, the transcriptional speed, pausing properties of the RNA polymerase, the effect of the transcriptional complex and associated factors, and the effect of RNA-binding proteins. Identifying strategic pause sites can reveal insights on the folding pathway of the nascent transcript. Structural mapping of the paused transcription complexes identifies important folding intermediates along these pathways. Oligohybridization assays and the appearance of the catalytic activity of a ribozyme either in trans or in cis can be used to monitor cotranscriptional folding under a wide range of conditions. In our laboratory, these methodologies have been applied to study the folding of three highly conserved RNAs (RNase P, SRP, and tmRNA), several circularly permuted forms of a bacterial RNase P RNA, a riboswitch (thiM), and an aptamer-activated ribozyme (glmS).

Introduction

The Mg²⁺-initiated refolding of fully synthesized RNA transcripts has been the primary experimental method used to investigate RNA folding pathways (Draper et al., 2005, Misra et al., 2003, Sosnick and Pan, 2003, Treiber and Williamson, 2001, Woodson, 2002, Woodson, 2005). These studies have shown that many RNAs fold through a rugged landscape kinetically dominated by the formation of and the subsequent escape from long-lived folding intermediates. Due to the small number of distinct nucleotide bases and the relative simplicity of Watson–Crick base pairing, RNA molecules exhibit a strong propensity to form non-native structures. Because of the thermodynamic strength of the Watson–Crick base pairs formed, these structures are often quite stable. The likelihood of forming long-lived folding intermediates grows as RNAs increase in size.

In vivo analyses of RNA folding have often revealed a different picture compared to in vitro studies performed on the same RNA molecule. The Tetrahymena group I intron is a widely studied model system for RNA folding (Cech and Bass, 1986, Doudna and Cech, 2002, Treiber and Williamson, 2001, Woodson, 2002). It is located in the gene for the large ribosomal subunit. In vivo, the half-life of this pre-rRNA has been measured at ~ 2 s (Brehm and Cech, 1983). However, when studied in vitro, this group I intron spliced at a rate ~ 20–50 times slower (Treiber and Williamson, 2001, Woodson, 2002). Presumably, when transcribed in vivo, this RNA manages to avoid the long-lived, nonfunctional structures which dominate its folding pathway in vitro.

Cotranscriptional RNA folding in vivo differs from Mg²⁺-initiated refolding in several aspects. RNA has an inherent 5′- to 3′-polarity. During transcription, the 5′-portion of the nascent transcript emerges from the polymerase before its 3′-region. Therefore, the upstream region can begin folding at an earlier time. Factors influencing this timing window and, subsequently, the structural formation of the upstream regions include the transcriptional speed of the RNA polymerase and transcriptional pausing. Cotranscriptional folding studies replicate the 5′- to 3′-polarity of the RNA transcript and can account for the properties of the RNA polymerase and its various transcription factors (Pan and Sosnick, 2006). As such, they provide better models for the in vivo folding pathways of RNA (Al-Hashimi and Walter, 2008, Pan and Sosnick, 2006).

This chapter describes several techniques to study cotranscriptional folding of RNA. Some methods are useful only for particular RNAs while others are more widely applicable. Examples are provided in which these techniques have been used to investigate the folding of several noncoding RNAs during transcription. They include three highly conserved noncoding RNAs (RNase P, SRP, and tmRNA), several circularly permuted forms of a bacterial RNase P RNA, a riboswitch (thiM), and an aptamer-activated ribozyme (glmS). Through the study of cotranscriptional folding, it is shown how the process of transcription, the properties of the polymerase (transcriptional pausing in particular), and the presence of additional factors in the cellular environment can influence the RNA folding pathway.

Section snippets

Protocol 1: Determination of Transcriptional Pause Sites

RNA polymerases do not transcribe their nascent transcripts at a uniform rate. Rather they have been found to pause at specific sequence or structure-dependent locations (Artsimovitch and Landick, 2000). At these pause sites, the elongation rate of the polymerase slows dramatically. Transcriptional pausing can influence RNA folding by increasing the time window at strategic locations so that upstream regions of the transcript can form beneficial native or non-native interactions. Pausing may

Protocol 2: Structural Mapping of Paused Complexes

Understanding how transcriptional pausing affects the folding of the nascent transcript requires the structural characterization of the RNA in the paused complex. Because these “truncated” RNAs lack significant portions of their full-length versions, the conformation of these nascent RNA transcripts most likely contain secondary and tertiary structures absent from the native conformation. Structural analyses of the paused complexes can shed light on these structures as intermediates in

Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization

Oligonucleotide hybridization with RNase H cleavage has been used to analyze the Mg²⁺-initiated refolding pathways of the group I intron and RNase P (Treiber and Williamson, 2000, Zarrinkar and Williamson, 1994, Zarrinkar et al., 1996). Briefly, as RNA folds into its native conformation, its secondary structures become more protected against hybridization to complementary DNA probes. The rates at which different regions of the RNA become protected can provide site-specific information on the

Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity

RNase P is a highly conserved ribozyme in both prokaryotes and eukaryotes. Its primary function is to generate the mature 5′-end of tRNAs through an endonucleolytic cleavage reaction (Altman and Kirsebom, 1999, Frank and Pace, 1998). In its native conformation, bacterial RNase P is catalytically active against precursor tRNAs. This can be used to investigate the cotranscriptional folding of this ribozyme. Of note, the following protocol may be modified for any trans-acting ribozyme as long as

Protocol 5: The Folding of Self-Cleaving RNAs During Transcription

Many catalytic RNAs are characterized by their ability to self-cleave at specific locations upon folding into their native conformations (Been, 2006, Doherty and Doudna, 2000, Doherty and Doudna, 2001, Fedor, 2000, Fedor and Williamson, 2005, Long and Uhlenbeck, 1993). This fact can be exploited to study the cotranscriptional folding of these RNAs. In vivo folding during transcription studies have primarily focused on the group I intron (Emerick and Woodson, 1993, Hagen and Cech, 1999, Jackson

Additional Methodologies

Several techniques, not described in detail above, have also been used to investigate the cotranscriptional folding of RNA. These include native gel electrophoresis (Heilman-Miller and Woodson, 2003), temperature-gradient gel electrophoresis (Repsilber et al., 1999), single-molecule techniques (Aleman et al., 2008, Dalal et al., 2006, Greenleaf et al., 2008), and computational approaches (Geis et al., 2008, Isambert and Siggia, 2000, Meyer and Miklos, 2004, Shapiro et al., 2006, Xayaphoummine

The folding of B. subtilis P RNA during transcription by T7 RNA polymerase (Pan et al., 1999)

The appearance of catalytic activity against pre-tRNA was used to assay the folding of B. subtilis P RNA. The folding of this ribozyme when transcribed by T7 RNA polymerase was compared to its Mg²⁺-initiated refolding. The cotranscriptional folding of B. subtilis P RNA entailed a pathway in which its C-domain folded approximately fourfold faster than its S-domain. This produced an experimentally observable folding intermediate that was catalytically active against the selected substrate but

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Riboswitches are an outstanding example of genetic regulation mediated by RNA conformational switching. In these non-coding RNA elements, the occupancy status of a ligand-binding domain governs the mRNA’s decision to form one of two mutually exclusive structures in the downstream expression platform. Temporal constraints upon the function of many riboswitches, requiring folding of complex architectures and conformational switching in a limited co-transcriptional timeframe, make them ideal model systems for studying these processes. In this review, we focus on the mechanism of ligand-directed conformational changes in one of the most widely distributed riboswitches in bacteria: the cobalamin family. We describe the architectural features of cobalamin riboswitches whose structures have been determined by x-ray crystallography, which suggest a direct physical role of cobalamin in effecting the regulatory switch. Next, we discuss a series of experimental approaches applied to several model cobalamin riboswitches that interrogate these structural models. As folding is central to riboswitch function, we consider the differences in folding landscapes experienced by RNAs that are produced in vitro and those that are allowed to fold co-transcriptionally. Finally, we highlight a set of studies that reveal the difficulties of studying cobalamin riboswitches outside the context of transcription and that co-transcriptional approaches are essential for developing a more accurate picture of their structure–function relationships in these switches. This understanding will be essential for future advancements in the use of small-molecule guided RNA switches in a range of applications such as biosensors, RNA imaging tools, and nucleic acid-based therapies.
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Chapter 9 - RNA Folding During Transcription: Protocols and Studies

Abstract

Introduction

Section snippets

Protocol 1: Determination of Transcriptional Pause Sites

Protocol 2: Structural Mapping of Paused Complexes

Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization

Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity

Protocol 5: The Folding of Self-Cleaving RNAs During Transcription

Additional Methodologies

The folding of B. subtilis P RNA during transcription by T7 RNA polymerase (Pan et al., 1999)

Curr. Opin. Struct. Biol.

Curr. Opin. Chem. Biol.

Protein Expr. Purif.

Chem. Biol.

Mol. Cell

J. Mol. Biol.

J. Mol. Biol.

Structure

J. Mol. Biol.

J. Mol. Biol.

Cell

Methods

Methods Enzymol.

Mol. Cell

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Graph. Model.

Curr. Opin. Struct. Biol.

J. Biol. Chem.

Methods Enzymol.

Curr. Opin. Struct. Biol.

Mol. Cell

Curr. Opin. Chem. Biol.

Ribonuclease P: An enzyme with a catalytic RNA subunit

Adv. Enzymol. Relat. Areas Mol. Biol.

Ribonuclease P

Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals

Proc. Natl. Acad. Sci. USA

RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro

J. Bacteriol.

HDV ribozymes

Curr. Top. Microbiol. Immunol.

Fate of an intervening sequence ribonucleic acid: Excision and cyclization of the Tetrahymena ribosomal ribonucleic acid intervening sequence in vivo

Biochemistry

Biological catalysis by RNA

Annu. Rev. Biochem.

Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor

J. Bacteriol.

Ribozyme structures and mechanisms

Annu. Rev. Biochem.

Ribozyme structures and mechanisms

Annu. Rev. Biophys. Biomol. Struct.

The chemical repertoire of natural ribozymes

Nature

Ions and RNA folding

Annu. Rev. Biophys. Biomol. Struct.

Self-splicing of the Tetrahymena pre-rRNA is decreased by misfolding during transcription

Biochemistry

The catalytic diversity of RNAs

Nat. Rev. Mol. Cell Biol.

Ribonuclease P: Unity and diversity in a tRNA processing ribozyme

Annu. Rev. Biochem.

Direct observation of hierarchical folding in single riboswitch aptamers

Science

Catalytic activity of an RNA molecule prepared by transcription in vitro

Science