Toeprint Assays for Detecting RNA Structure and Protein–RNA Interactions

Abstract

Toeprint assays are primer extension inhibition assays that can detect the 3′ end of an RNA secondary structure, the position of a bound RNA binding protein, as well as the position of a bound 30S ribosomal subunit or a stalled ribosome. Here we describe how this assay was used to identify an RNA hairpin that sequesters a Shine–Dalgarno sequence, how the RNA-binding protein CsrA can alter RNA structure and affect 30S ribosomal subunit binding, and how the macrolide antibiotic tylosin can induce ribosome stalling.

1. Introduction

The toeprint assay, also known as primer extension inhibition, is a method used in molecular biology for detecting RNA structure and protein–RNA interactions. This method is useful to identify the 3′ end of an RNA secondary structure, changes in RNA structure after interaction with an RNA-binding protein, and for determining the position of the 3′ edge of a bound protein [1, 2]. This assay can also provide mechanistic insight into the regulation of translation initiation by determining the position of the 3′ edge of a bound 30S ribosomal subunit in the presence or absence of an RNA-binding protein; bacterial 30S ribosomal subunits typically give an tRNA^fMet-dependent toeprint band 16 nt downstream from the A of the AUG initiation codon [1–3]. Toeprint assays have also been used in coupled transcription-translation reactions to identify the position of antibiotic-induced ribosome stalling during polypeptide synthesis [4, 5]. It is important to empirically identify an appropriate buffer and temperature that is compatible with protein function, ribosome function, and reverse transcriptase activity. It is absolutely critical to titrate reverse transcriptase to determine the optimal amount of enzyme that will allow you to identify the various molecular interactions. Too much reverse transcriptase can lead to full-size primer extension products and the absence of reverse transcription stops at the position(s) of bound proteins, ribosomes, and/or RNA structure. The utility of the toeprint assay is exemplified by demonstrating how binding of Escherichia coli CsrA protein disrupts an RNA structure that sequesters the ymdA Shine–Dalgarno (SD) sequence, how E. coli CsrA can affect ribosome binding, and how the antibiotic tylosin induces ribosome stalling during translation of the Bacillus subtilis tlrB leader peptide.

2. Materials

RNase-free conditions must be maintained throughout the procedure. All glassware, spatulas, and stir bars used for preparing solutions should be baked in an oven at 250 °C for 5 h to destroy RNases. Prepare all solutions with ultrapure water. All solutions that come into contact with RNA should be treated with diethylpyrocarbonate (DEPC) followed by autoclaving (see Note 1).

2.1. DNA Template for In Vitro Transcription (see Note 2)

1. Vent DNA polymerase.

2. 20 μM primers. Forward primer contains the T7 RNA polymerase (RNAP) promoter sequence in addition to complementarity to the target sequence.

3. Plasmid DNA or chromosomal DNA template.

4. Thermocycler.

5. PCR purification kit.

2.2. RNA Template Preparation (see Note 1)

1. DEPC-treated water: Add 1 mL of DEPC to 0.5 L of distilled water in a 1 L bottle. Mix, incubate overnight at room temperature in a hood, and autoclave for 30 min the next day. Store at room temperature.

2. In vitro transcription is carried out using the MEGAscript T7 or RNAMaxx transcription kit according to the manufacturer’s specifications.

3. 5X TBE buffer for gel purification: Mix 465 mM Tris, 445 mM boric acid, 10 mM EDTA, pH 8.3. Weigh 54 g of Tris base, 27.5 g of boric acid, and 3.7 g of EDTA disodium salt in a 1 L glass beaker containing a stir bar. Add water to a volume of ~600 mL. Mix on a magnetic stirrer. Add water to a final volume of 1 L. pH does not require any adjustment. Filter and store at room temperature.

4. 6% polyacrylamide gel solution: Mix 75 mL of 40% acrylamide: bisacrylamide (19:1), 249.25 g urea, 100 mL 5X TBE buffer, and add water to a final volume of 500 mL. Filter and store at 4 °C. Mix 12 mL of this solution with 12 μL of TEMED and 120 μL of fresh 10% ammonium persulfate. Cast gels using 10 × 10 cm gel plates with 0.5 mm spacers. Insert a 10 well comb. Allow the gel to polymerize for at least 30 min.

5. 2X RNA loading buffer (stop solution): For 10 mL mix 74.4 mg EDTA disodium salt, 48.4 mg Tris base, 10 mg sodium dodecyl sulfate (SDS), 3 mg bromophenol blue, and 3 mg xylene cyanol. Dissolve in 9.5 mL of formamide and store at −20 °C.

6. Shortwave UV light (254 nm) and a fluor-coated thin layer chromatography (TLC) plate for visualizing nucleic acids separated on polyacrylamide/urea gels.

7. Baked razor blades.

8. RNA elution buffer: 1.92 g ammonium acetate (0.5 M), 0.093 g EDTA disodium salt (5 mM), 0.1 g SDS (0.2%), and water to 50 mL. Treat solution with 50 μL DEPC and autoclave 30 min the next day.

9. Glycogen solution (5 mg/mL).

10. Phenol for RNA.

11. Chloroform.

12. 3 M sodium acetate, pH 5.2: Add 24.6 g of sodium acetate (anhydrous) to 70 mL distilled water. Dissolve the sodium acetate and adjust the pH to 5.2 with glacial acetic acid. Add water to a final volume of 100 mL.

13. 100% and 70% ethanol.

14. 100 mM dithiothreitol (DTT): dissolve 154 mg DTT in 10 mL of DEPC-treated water. Store at −20 °C.

15. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. Mix 0.5 mL of 2 M Tris-HCl, pH 7.5, 0.2 mL of 0.5 M EDTA, and 99.3 mL of DEPC-treated water.

2.3. 5′ End-Labeled Primer

1. 18–25 nt DNA oligonucleotide with a melting temperature (Tm) of about 55 °C. The oligo should be complementary to a region near the 3′ end of the transcript that will be used in the toeprint reaction. The oligonucleotide should hybridize between 60 and 200 nt downstream of the expected toeprint position.

2. [γ−³²P] ATP (6000 Ci/mmol, 150 mCi/mL).

3. T4 polynucleotide kinase.

4. Mini quick spin oligo columns.

2.4. Toeprint Reaction

1. Reverse transcriptase (see Note 3).

2. 5X toeprint reaction buffer: 200 mM Tris-HCl, pH 8.0, 1 M KCl, 20 mM MgCl₂, 5 mM DTT. For 1 mL mix 0.1 mL 2 M Tris-HCl, pH 8.0, 0.25 mL 4 M KCl, 0.04 mL 0.5 M MgCl₂, 50 μL 100 mM DTT, and 560 μl of water. Store at −20 °C (see Note 3).

3. dNTP mixture.

4. RNasin RNase inhibitor.

5. 100 mM DTT.

6. 10 mg/mL BSA (Promega, NEB).

7. Protein dilution buffer (PDB): 10 mM Tris-HCl, pH 8, 1 mM DTT, 10% glycerol. Mix 10 μL 1M Tris, pH 8.0, 10 μL 100 mM DTT, 100 μL 100% glycerol, and 880 μL water. Store at −20 °C.

8. tRNA^fMet. We have been unable to identify a commercial source for this reagent.

9. 30S ribosomal subunits [6].

10. Yeast RNA. Dissolve 10 mg yeast RNA in 1 mL of DEPC-treated water. Store at −20 °C.

11. PURExpress in vitro protein synthesis kit.

2.5. Sequencing Gel

1. Sequenase version 2.0 DNA sequencing kit.

2. Gel casting system for sequencing gels.

3. Sigmacoat.

4. Whatman filter paper and plastic wrap.

3. Methods

3.1. DNA Template Containing a T7 RNAP Promoter

1. PCR reaction in 50 μL: 1 μL of Vent DNA polymerase, 5 μL of each 20 μM primers (forward primer contains the T7 RNAP promoter sequence), 5 μL of 10X buffer, 10–20 ng of plasmid DNA template or 100–300 ng of chromosomal DNA template, 1 μL of 100 mM MgSO₄ (supplied with Vent). 30–35 cycles with an annealing temperature 5 °C less than the melting temperature (Tm) of the primers, and extension at 72 °C for 2 min per kb. Examine a 5 μL aliquot of each reaction on an agarose gel.

2. Purify the PCR product with a PCR purification kit according to the manufacturer-provided manual.

3.2. RNA Preparation and Purification

1. Synthesize RNA with the MEGAscript T7 or RNAMaxx transcription kit. Use about 300 ng of the purified PCR product or 1 μg plasmid as a DNA template for each reaction according to the manufacturer’s specifications.

2. Make 6% polyacrylamide gel solution with 8.3 M urea. Cast gel using 10 × 10 cm gel plates with 0.5 mm spacers. Insert a 10 well comb. Allow the gel to polymerize for at least 30 min. Transfer gel to a vertical gel running apparatus and fill the buffer reservoirs with 1X TBE buffer. Prerun the gel at 5 Watts for 10 min. Mix the RNA sample with the same volume of 2X loading buffer. Run gel at 3–5 Watts (see Note 4).

3. After electrophoresis, transfer gel from plates to plastic wrap. Cover the gel with a second sheet of plastic wrap. In a darkened room, place the plastic-wrapped gel on top of the fluor-coated TLC plate. Visualize nucleic acid bands by shining a hand-held UV light source (254 nm; shortwave) on the surface of the gel. Nucleic acid will appear as dark purple bands (see Note 5).

4. Mark the band to be removed. Cut out the band with a baked razor blade. Place the gel slice into a 2 mL microfuge tube with 300 μL of RNA elution buffer. Incubate at 37 °C overnight with shaking. The next day, centrifuge the tube at maximum speed for 5 min and then transfer the liquid to a new tube. Add glycogen to 50 μg/mL as a carrier for RNA precipitation. Add an equal volume of phenol and chloroform, vortex for 1–2 min, and then centrifuge for 5 min at maximum speed. Transfer the top aqueous layer to a new microfuge tube. Add 1/10 vol of 3 M sodium acetate, pH 5.3, and 2 vol of 100% ethanol. Incubate at −20 °C for 2 h and then pellet the RNA by centrifuging at 4 °C for 30 min at maximum speed. Wash the RNA pellet with 70% ethanol. Centrifuge at maximum speed for 5 min. Dry the RNA pellet and then dissolve in 30 μL of TE buffer. Determine the RNA concentration and then dilute to 300 nM. Store at −80 °C.

3.3. Preparation of 5′ End-Labeled Primer

1. For a 10 μl reaction, mix 1 μL of 25 μM DNA oligonucleotide, 1 μL of 10X T4 polynucleotide kinase buffer, 2 μL of 25 μM [γ−³²P] ATP, 5 μL water, and then 1 μL (10 U) of T4 polynucleotide kinase.

2. Mix and incubate at 37 °C for 60 min.

3. Purify labeled primer using a mini quick spin oligo column. Centrifuge the column for 1 min at 3000 rpm, add 300 μL TE buffer and centrifuge for 2 min at the same speed.

4. Add 40 μL TE buffer to the 10 μL labeling reaction, then load onto the center of the column matrix, and centrifuge for 4 min.

5. Determine the concentration of the labeled primer and then dilute to 300 nM. Store at −80 °C.

3.4. Sequencing Ladder

1. Mix 1 μL of 1 μM plasmid DNA (see Note 2) or 1 μL of 1 μM PCR fragment and 3.33 μL of 300 nM of 5′ end-labeled oligonucleotide primer (see Subheading 3.3). Add 5.7 μL of water and then mix. Add 10 μL freshly prepared 2 N NaOH and incubate at 37 °C for 10 min. Add 2 μl 5 mg/mL glycogen, then add 6.7 μL 3 M sodium acetate, pH 5.2, and then add 175 μL of 100% ethanol. Mix and incubate at −20 °C for at least 1 h. Centrifuge at 4 °C for 30 min at maximum speed, wash with 70% ethanol, and then dry pellet. Dissolve pellet in 20 μL of 1X sequenase buffer (the buffer supplied in the sequenase kit is 5X).

2. Mix 4 μL of 100 mM DTT, 13.4 μL water, and 1 μL sequenase. Then add 11 μL of this mixture to the plasmid and primer in sequenase buffer. Transfer 5 μL of each ddNTP from the sequenase kit to separate tubes. Prewarm tubes at 37 °C, then add 7 μL of the sequenase-containing mixture and incubate at 42 °C for 5 min. Stop the reaction by adding 12 μl of 2X RNA loading buffer. Transfer 3 μL of each reaction to a new tube and incubate at 95 °C for 5 min prior to loading onto a sequencing gel.

3.5. Toeprint Reaction with E. coli CsrA and ymdA RNA to Demonstrate How Protein-Binding Can Alter RNA Structure

1. Hybridization reaction for 1 toeprint reaction (scale up as necessary): Prepare hybridization reaction by mixing 1 μL of 150 nM gel-purified RNA (see Subheading 3.2) and 1 μL of 150 nM of the 5′-end-labeled DNA primer (see Subheading 3.3) that is complementary to the 3′ end of the RNA. Incubate at 85 °C for 3 min, and then slowly cool down on the bench to room temperature (~10 min).

2. Reverse transcriptase mixture sufficient for 1 toeprint reaction (scale up as necessary): Mix 1 μL 10X SuperScript III reverse transcriptase buffer, 0.4 μL of 10 mM dNTP solution, 1 μL of 100 mM DTT, 1 μL of 2 μg/μL yeast RNA, 0.1 μL of RNasin, 0.2 μL 10 mg/mL BSA, and 2.3 μL water.

3. For each toeprint reaction mixture (10 μL) combine 2 μL of the hybridization mixture, 2 μL of 7.5 μM CsrA protein (1.5 μM final concentration) or the same volume of PDB, and 6 μL of the reverse transcriptase mixture.

4. Incubate at 37 °C for 20 min to allow CsrA–RNA complex formation.

5. Add 1 μL of SuperScript III reverse transcriptase (diluted in PDB) and continue incubation at 37 °C for 15 min (see Note 6).

6. Add an equal volume (11 μL) of 2X stop solution. Heat a 3 μL aliquot of each toeprint reaction and sequencing ladder samples at 95 °C for 5 min. Load onto a 6% sequencing gel that has been prerun for 20 min at 80 Watts. Continue running at 80 watts after loading the samples.

7. Remove gel from glass plates and transfer to Whatman filter paper, cover with plastic wrap, and dry on a gel dryer at 80 °C for 30 min. Place completely dried gel in a phosphor imager cassette overnight or longer.

8. Compare lanes with and without protein to identify differences in the banding pattern and determine the precise positions using the sequencing ladder.

9. In the absence of bound CsrA, two adjacent RNA structure-dependent toeprint bands were identified at positions −11A and −12G (see Fig. 1a, lane 2). These bands are located at the base of the SD-sequestering hairpin, indicating that this hairpin forms in the absence of bound CsrA (see Fig. 1b). Two adjacent CsrA-dependent toeprints were observed at nucleotides −24C and −25A (see Fig. 1a, lane 3), which are at the 3′ edge of CsrA bound at binding site 2 (BS2). The absence of the RNA structural toeprint bands in the presence of CsrA indicates that bound CsrA disrupts the SD-sequestering hairpin.

Fig. 1.

RNA structure, CsrA, and ribosome toeprint analysis. (a) CsrA and 30S ribosome toeprint analysis of ymdA leader RNA. Bound CsrA facilitates 30S ribosomal subunit binding by disrupting the ymdA SD-sequestering hairpin. The presence of CsrA, tRNA^fMet, 30S ribosomal subunit (30S Rib), and/or reverse transcriptase (SSIII RT) is indicated above each lane. Positions of CsrA (CsrA), 30S ribosomal subunit (30S Rib), and SD-sequestering hairpin (SD hairpin) toeprints are indicated with arrows. Sequencing lanes A, C, G, and U are marked, and lane numbers are shown at the bottom of the gel. Numbering is with respect to the start of ymdA translation. (b) Model for CsrA binding to the ymdA leader region. GGA motifs of CsrA binding sites 1 and 2 (BS1 and BS2) are in red. The SD sequence and ymdA translational start codon (Met) are marked. (This figure was adapted from Ref. 2. Copyright © American Society for Microbiology, mBio 11:e00849–20 (2020). DOI: 10.1128/mBio.00849-20)

3.6. 30S Ribosomal Toeprint with E. coli ymdA RNA to Demonstrate How CsrA Can Affect Ribosome Binding

1. Prepare hybridization mixture (see Subheading 3.5, step 1).

2. Prepare reverse transcriptase mixture (see Subheading 3.5, step 2). Instead of adding 2.3 μL water, only add 0.3 μL, which will reserve 2 μL for adding 30S ribosomal subunits and/or tRNA^fMet.

3. For each toeprint reaction mixture (10 μL), combine 2 μL of the hybridization mixture (see Subheading 3.5, step 1) and 4 μL of the reverse transcriptase mixture (see Subheading 3.5, step 2). Then add 1 μL of 100 μM tRNA^fMet (10 μM final concentration), 1 μL of 50 μM 30S ribosomal subunits (5 μM final concentration), and/or 2 μL of 7.5 μM CsrA (1.5 μM final concentration). Bring the total volume to 10 μL by adding PDB for reactions without CsrA or ribosomes and/or TE buffer for reactions without tRNA^fMet as necessary (see Notes 7 and 8).

   • 3a.
     Add 2 μL of 7.5 μM CsrA and incubate at 37 °C for 20 min (see Note 7).
   • 3b.
     Add 1 μL of 100 μM tRNA^fMet.
   • 3c.
     Add 1 μL of 100 μM tRNA^fMet and 1 μL of 50 μM 30S ribosomal subunits (see Note 8).
   • 3d.
     Add 2 μL of 7.5 μM CsrA and incubate at 37 °C for 20 min. Then add 1 μL of 100 μM tRNA^fMet.
   • 3e.
     Add 2 μL of 7.5 μM CsrA and incubate at 37 °C for 20 min. Then add 1 μL of 100 μM tRNA^fMet and 1 μL of 50 μM 30S ribosomal subunits.

4. Then follow the procedure as written in Subheading 3.5, steps 5–8.

5. The addition of only tRNA^fMet had no effect on the banding pattern (see Fig. 1a, compare lanes 2 and 4). The addition of tRNA^fMet and 30S ribosomal subunits resulted in a smear near the base of the SD-sequestering hairpin but no toeprint band downstream of the start codon (see Fig. 1, lane 5). When CsrA was added prior to the addition of tRNA^fMet, the CsrA toeprint bands were observed (see Fig. 1a, compare lanes 3 and 6). When CsrA was added prior to the addition of tRNA^fMet and 30S ribosomal subunits, two ribosomal toeprint bands were observed at +16U and +18A (see Fig. 1a, compare lanes 5 and 7). +16U is the expected position of a 30S ribosomal subunit toeprint. These results indicate that bound CsrA promotes 30S ribosomal subunit binding by destabilizing the ymdA SD-sequestering hairpin.

3.7. Toeprint Analysis of Tylosin-induced Ribosomal Stalling in the tlrB Leader Using PURExpress

1. Prepare DNA template with T7 promoter by PCR (see Subheading 3.1). Dilute template to 250 ng/μL.

2. Prepare 5′ end-labeled primer (see Subheading 3.3).

3. Transcription-translation mixture for 1 toeprint reaction (scale up as necessary): Mix 0.2 μL DNA template, 1.1 μL water, 2 μL solution A from the PURExpress kit, and 1.5 μL solution B from the PURExpress kit. Add 0.5 μL of tylosin at various concentrations (add water instead of tylosin as a negative control). Incubate at 37 °C for 1 h.

4. Hybridization mixture for 1 toeprint reaction (scale up as necessary): Mix 3 μL of 5X first strand (FS) buffer, 1.5 μL 100 mM DTT, 1.5 μL 2.5 mM dNTP, 0.375 μL RNasin, 1.125 μL water, 1 μL 5′ end-labeled primer (see Subheading 3.3).

5. Hybridization reaction for 1 toeprint reaction (scale up as necessary): Combine the 5.3 μL transcription-translational mixture (± tylosin) with 8.5 μL hybridization mixture. Incubate at 55 °C for 3 min. Chill on ice.

6. Add 0.5–2 μL of SuperScript III reverse transcriptase. Incubate at 37 °C for 1 h (see Note 6).

7. Then follow Subheading 3.5, steps 6–8.

8. A tylosin-dependent toeprint band was identified at position U74 with the WT tlrB leader template (see Fig. 2a, lane 2). This toeprint band was not observed in the RYR to AYA mutant template in which the tylosin-dependent ribosome stall motif was disrupted in the tlrB leader peptide (see Fig. 2a, lane 4 and 2b).

Fig. 2.

Tylosin-dependent ribosome stalling in the tlrB leader peptide. (a) Toeprint analysis of tylosin-induced ribosome stalling during translation of the leader peptide using wild type (WT) and AYA mutant templates. The toeprint (TP) identified with the WT template in the presence of tylosin (+) is marked. Sequencing lanes A, C, G, and U are marked. The PURExpress kit containing T7 RNAP and E. coli ribosomes was used for this analysis. (b) tlrB leader region covered by the ribosome when tylosin induces stalling. Positions of the toeprint, and the ribosome peptidyl (P) and aminoacyl (A) sites are shown. The leader peptide coding sequence, and the leader peptide and tlrB SD sequences are shown. Numbering is with respect to start of tlrB transcription. (This figure was adapted from Ref. 5. Copyright © American Society for Microbiology, mBio 10:e02665–19 (2019). DOI: 10.1128/mBio.02665-19)

4. Notes

1. Maintaining RNase-free conditions is a vital consideration. Gloves should be worn at all times. Avoid using pipeters that have been used for procedures containing RNase A (e.g., plasmid purification). DEPC must be destroyed by autoclaving.

2. The use of a small plasmid vector is recommended to clone genes of interest (e.g., pTZ19R) to improve the generation of DNA templates via PCR and for DNA sequencing ladders. The concentration of Mg²⁺ in PCR using Vent DNA polymerase should be adjusted according to the manufacturer’s suggestions.

3. Perform gel mobility assays to identify an appropriate buffer for toeprint reactions [7]. The buffer must be suitable for protein binding and reverse transcriptase activity. For example, we used 5X toeprint buffer (see Subheading 2.4, step 2) for B. subtilis TRAP protein toeprints (1) and avian myeloblastosis virus (AMV) or moloney murine leukemia virus (MMLV) reverse transcriptases (see Subheading 2.4, step 1). Alternatively, we used SuperScript III buffer and SuperScript III reverse transcriptase for E. coli CsrA toeprints (2).

4. 100 nt RNA runs approximately at the same place as xylene cyanol.

5. The xylene cyanol and bromophenol blue dyes may appear as dark bands during UV shadowing and are sometimes mistaken for nucleic acid. We recommend running an extra lane of loading buffer alone to easily distinguish between these dyes and a shadow caused by RNA.

6. It is vital to titrate RT to identify an appropriate amount of enzyme that maximizes the amount of toeprint signal at the expense of full-length products.

7. Subheading 3.6, steps 3a–3e are separate reactions containing various combinations of CsrA, tRNA^fMet, and/or 30S ribosomal subunits. Always add CsrA or other RNA-binding protein first to allow protein–RNA complex formation. Then add tRNA^fMet and/or 30S ribosomal subunits.

8. Ribosomal subunits need to be activated by incubating at 37 °C for 15 min before adding to the toeprint reaction.