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Natural RNA sensors of small molecules (a.k.a. riboswitches) regulate numerous metabolic genes. In bacteria, these RNA elements control transcription termination and translation initiation by changing the folding pathway of nascent RNA upon direct binding of a metabolite. To identify and study riboswitches we used in vitro reconstituted solid-phase transcription elongation/termination system. This approach allows for direct monitoring ligand binding and riboswitch functioning, establishing the working concentration of a ligand as a function of RNA polymerase speed, and also probing RNA structure of the riboswitch. Using this system we have been able to identify and characterize first several riboswitches including those involved in vitamin biosynthesis and sulfur metabolism. The system can be utilized to facilitate biochemical studies of riboswitches in general, i.e. to simplify analysis of riboswitches that are not necessarily involved in transcriptional control.
In the last several years, riboswitches have become a paradigm of the RNA-driven regulation of gene expression (1–5). These non-coding stretches of RNA adopt an alternative conformation upon direct binding of a small molecular ligand to activate or repress cognate genes. In bacteria, riboswitches are ubiquitous and control over 3% of all metabolic genes. They usually reside in the 5′ untranslated regions (UTR) and consist of two coupled elements: evolutionary conserved sensor domain, which directly binds the ligand, and a variable expression platform that transmits the signal to the gene expression machinery such as RNA polymerase or a ribosome (1, 2). Depending on its design, a riboswitch either prevents or permits the formation of an RNA attenuator that in turn dictates whether a gene is expressed or not. The attenuator functions as sequester of the ribosome-binding site or intrinsic terminator positioned in front of an operon or both. A few riboswitches that have been found in fungi and plants work at the level of alternative RNA splicing (6–8). Since the sensor domain is highly conserved within each family of riboswitches, it can be placed within a context of a standard transcription termination expression platform to benefit from a simple and quantitative in vitro readout system. Such a strategy also takes full advantage of the solid-phase reconstituted transcription system for thorough biochemical analysis of riboswitch functioning. The system utilizes “walking” technology to obtain individual homogenous elongation complexes (EC) stalled at any desired position within the transcription unit (9). This allows for various types of RNA structural analysis (e.g. chemical and enzymatic probing, crosslinking, and real-time spectrometry) to be performed during transcription thus reflecting a natural situation of co-transcriptional RNA folding. The system also takes into account the kinetics of transcription elongation.
The common structure of the template for riboswitch studies is shown in Fig. 1. It consists of the A1 promoter of bacteriophage T7, adapter sequence, the sensor domain of a riboswitch, and an intrinsic transcription terminator to be coupled with a riboswitch of choice. T7A1 is one of the strongest known promoters to support in vitro transcription by RNA polymerase (RNAP) from Gram-negative or Gram-positive bacteria, including E.coli and B.subtilis. It works within a broad range of temperatures and salt concentrations and supports multi-round transcription in vitro without additional factors. The adapter sequence is a common initial transcribed sequence (ITS), which allows for one step preparation of the homogenous highly radiolabeled elongation complex stalled at position +20°C (EC20). The terminator hairpin sequence should be designed to match the structure of the sensor domain of a riboswitch. The 3′ proximal part of the terminator hairpin stem is variable and should be complementary to the antiterminator part of the expression platform. It is followed by a stretch of 8–9 thymidines (T-stretch) and GC-rich “front element” of the terminator. The latter part improves the termination efficiency (12). The following procedure exemplifies the walking technique for the T7 A1 promoter template with the initial transcribed sequence: ATCGAGAGGG10 ACACGGCGAA20 TAGCCATCCC30 AAT33.
T7A1 promoter containing linear DNA templates can be easily produced by PCR and purified in a relatively large concentration (e.g. 1 pmol/μL). At least two fold molar excess of DNA over RNAP is recommended to achieve the maximum yield of the initial EC.
The procedure described below is optimized for E. coli RL 721 strain (10), a KmR derivative of JC7623 strain (13). This bacterial strain carries a chromosomal copy of the rpoC gene encoding a β′ subunit of RNA polymerase with the His-tag at its COOH-terminus. The use of this strain guarantees that all purified RNAP would be His-tagged, although essentially the same procedure could be applied to conventional E.coli and B.subtilis strains. The purification method described here yields ~1 mg of RNA polymerase σ70 (RNAPσ70) holoenzyme per 10 g of cells. The enzymatic activity of the holoenzyme is ~2–3 fold higher than that of a commercially available preparations of E.coli RNAP.
The principle of the walking reaction is that the initial EC immobilized onto a solid support undergoes rounds of washing to remove the unincorporated NTP substrates, followed by addition of the incomplete set of NTPs that allow transcription to proceed to the next DNA position corresponding to the first missing NTP.
Each component of the EC (RNAP, DNA, or RNA) can be tagged for walking. Biotin tag can be put at the 5′ terminus of the DNA strand (see below). For the His-tag, two types of metal-chelating beads are available: Ni++-NTA-agarose and TALON Co2+-Sepharose beads. The latter resin has less non-specific binding and requires less time and concentration of imidazole to elute the EC off the beads, if necessary. For the biotin tag, the best resin for walking is NeutrAvidine UltraLink from Pierce. A representative walking protocol, which is described here for the His-tagged β′ subunit of RNA polymerase, can be utilized with minimal changes for other tags (see Note 2).
Walking the EC far from the promoter (e.g. over 200 bp) may be laborious and time consuming. The yield of the final EC in this case can be decreased substantially due to partial loss of material (beads) during multiple washing steps and also due to partial loss of activity at certain (arrest) positions. To avoid these complications, a transcriptional roadblock can be used. The roadblock is a site-specific DNA binding protein that is able to stop the EC completely without termination at any distance from the promoter. The roadblock, which worked well in our hands is the mutant form of EcoRI restriction endonuclease, EcoRQ111 (14). EcoRQ111 can be removed from DNA without interfering with the EC by the high salt buffers (e.g. 500 mM KCl). The use of EcoRQ111 has an advantage over other site-specific DNA binding proteins in that it requires small changes in DNA for generating its binding site.
The following assay is designed for a quick assessment of the intrinsic termination efficiency as a function of riboswitch activity (Fig. 3, last two lanes).
To study the process in more detail, the assay can be modified to include walking and roadblocking steps. Once the initial immobilized EC is prepared as described above, it can be walked to any desired position along the riboswitch sequence. For example, to reach the sensor domain of the TPP-sensing riboswitch (thi-box) (16) at least four walking steps are needed (Fig. 3). Alternatively, the EcoRI site can be engineered to halt the EC at any distance within the riboswitch sequence. Using such walking techniques one can study the effect of co-transcriptional RNA folding on riboswitch functioning and also probe the RNA structure directly during elongation.
Many types of chemical and enzymatic RNA probing can be performed without releasing the EC from the solid support. The following methods of RNA probing have been used to monitor riboswitch conformational changes during transcription in response to ligand binding.
Major conformational changes in RNA can be readily detected by ribonuclease H (RNAse H) following the annealing of short (8–10 nt) antisense DNA oligos to the different part of the riboswitch nascent transcript (16, 17). RNAse H specifically recognizes RNA:DNA heteroduplexes and works in a standard TB.
Walking can be used for generating specifically modified RNA transcripts for structural analysis (12). Many different NTP analogs can be utilized by E. coli RNAP and incorporated into the nascent RNA at specific positions during walking, for example, within the sensor domain of the riboswitch. The cross-linkable NTP analogs (such as 4-thio-UTP and 6-thio-GTP) and fluorescent analogs (e.g. 2-aminopurinetriphosphate (2AP); 5-(fur-2-yl)UTP; or pyrollo-C) are particular useful to probe the local RNA conformational changes and interactions directly in the elongation complexes. For example, stacking interactions with the neighboring bases quench the 2AP fluorescence. Many NTP analogs and conditions for their incorporation are available from our laboratory upon request.
Direct RNA binding of a small molecule, which is intrinsically fluorescent (e.g. FMN) or carries a fluorescent tag that can be detected in real time during transcription by scanning spectrofluoremetry (16). The change of fluorescence during steady-state transcription is monitored on a Perkin-Elmer LS50B scanning fluorometer equipped with the quartz “submicrocuvette” with the 10 mm pathlength (see Note 4). For example, the formation of the riboswitch-FMN complex quenches FMN fluorescence due to photoinduced electron transfer from FMN to the aromatic rings of RNA bases.
This work was supported by the NIH grants R01 GM58750 and GM72814 (E.N.)
1Phenol and high temperature (>70°C) treatments affect the quality of DNA templates. Avoid phenol extraction or keep phenol treatment as brief as possible. To obtain DNA for biotin-tag-based walking, use for PCR the PAGE-purified forward DNA oligonucleotide carrying the 5′-biotin.
2Perform all reactions in siliconized Eppendorf plastic tubes. The standard round of washing, i.e. pelleting and resuspending the beads, includes 3–5 sec centrifugation in a table-top microcentrifuge, removal of supernatant leaving ~50 μL above the pellet, and resuspension in 1.5 mL of the appropriate transcription buffer.
3Commercially available NTPs are not pure enough to support walking on every DNA sequences. A read-through of certain positions due to small contamination in the NTP stocks is common. To avoid this problem, it is strongly recommended to purify original NTP stocks. The purification procedure has been described in detail (9).
4The method detects the effect of various conditions (e.g. the rate of elongation, salt concentration, etc.) on ligand binding during transcription. Spectroscopic detection can be coupled with the fast kinetic and walking techniques.