Name: Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function (Video) | JoVE Journal
Uploaded: 2017-06-21T12:30:00.000+00:00
Duration: 8 min 3 s
Description: Learn agarose gel retardation assays to analyze TcdA-tRNA interactions and characterize RNA-binding proteins. This method enables rapid, sequence-specific protein-RNA binding studies using minimal sample amounts.

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5 minutes

MCV is member of the CIB Intramural Program &#8220;Molecular Machines for Better Life&#8221; (MACBET). The research leading to these results has received funding from the Spanish Instituto de Salud Carlos III (PI12/01667 to MCV), the Spanish Ministerio de Econom&#237;a y Competitividad (CTQ2015-66206-C2-2-R and SAF2015-72961-EXP to MCV), the Regional Government of Madrid (S2010/BD-2316 to MCV), and the European Commission (Framework Programme 7 (FP7)) project ComplexINC (Contract No. 279039 to MCV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Caution: Consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in this protocol are toxic and corrosive. Use appropriate practices when performing the extraction of tRNA using phenol, including the use of a fume hood and personal protective equipment (safety glasses, gloves, lab coat, full-length pants, closed-toe shoes, etc.). This protocol uses a non-toxic nucleic acid stain. The use of alternative stains may require additional precautions and specialized disposal of the staining agent if toxic and/or carcinogenic (e.g., ethidium bromide).
1. Preparation of the Agarose Gel
<ol>
	<li>Weigh 2 g of agarose and add it to a glass bottle to prepare a 2% w/v agarose gel.</li>
	<li>Add 100 mL of 1x tris-borate-ethylenediaminetetraacetic acid (EDTA) (TBE) running buffer to the agarose-containing bottle. Melt by heating in a microwave. At 30 s intervals, swirl the contents to mix well and repeat this step until the agarose is completely dissolved. Allow the agarose solution cool down to approximately 50-60 &#176;C.</li>
	<li>Tape the open edges of a gel tray to create a mold, place an appropriate comb to create the wells and cast the molten agarose into it. Avoid bubble formation and let it solidify at room temperature.</li>
	<li>Once solidified, place the gel tray in an electrophoresis unit and fill it with TBE buffer until the gel is fully covered.</li>
</ol>
2. Preparation of the tRNA
<ol>
	<li>Express tRNA(UUU) in E. coli
	<ol>
		<li>In the morning, use a vial of frozen E. coli BL21(DE3) transformed with the expression vector pET23d-EcUUU2 (harbors the gene encoding E. coli tRNA(UUU)), to streak a low-salt Luria Broth (LB) plate supplemented with 100 &#181;g/mL ampicillin. Invert the plate and place it into a 37 &#176;C incubator until colonies appear (late afternoon).</li>
		<li>Inoculate one well-grown, single colony in 5 mL LB plus 100 &#181;g/mL ampicillin and grow overnight at 220 rpm at 37 &#176;C.</li>
		<li>The next morning, pellet the cells (6,500 x g for 10 min at 4 &#176;C), replace medium with 5 mL fresh LB plus 100 &#181;g/mL ampicillin and resuspend. Use this suspension to inoculate 200 mL LB plus 100 &#181;g/mL ampicillin. Grow for 5 h at 220 rpm at 37 &#176;C.</li>
		<li>When the optical density of the culture at 590 nm (OD590) has reached 0.6 - 0.8, add 1 mM IPTG to the culture to trigger the expression of the tRNA(UUU) gene. Incubate at 37 &#176;C for 3 h.</li>
		<li>Harvest the cells by centrifuging the culture at 6,500 x g for 10 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Lysis and extraction
	<ol>
		<li>Resuspend the cell pellet in 5 mL lysis buffer (0.3 M sodium acetate, 10 mM EDTA). From this point onward, used autoclaved buffers and keep all the tRNA-containing samples at 4 &#176;C to prevent tRNA degradation.</li>
		<li>Add 1 volume of acid phenol (pH 4.3) to the resuspended cell pellet in the fume hood. Mix for 1 min by inversion to lyse the cells and centrifuge at 10,000 x g (10 min, 4 &#176;C).</li>
		<li>Collect the upper aqueous phase containing the soluble tRNA(UUU) (and smaller concentrations of the other tRNA pools) using a pipette taking care not to aspirate the organic (bottom) phase. Discard the bottom layer and the pellet, which consists of the organic phase and cell debris, into an adequate container.</li>
		<li>Precipitate the tRNA by thoroughly mixing the soluble (aqueous) phase with 2.5 volume 100% v/v ethanol by inversion for 1 h at 4 &#176;C. Centrifuge the mixture at 15,000 x g (30 min, 4 &#176;C).</li>
		<li>Decant the supernatant carefully and resuspend the tRNA-rich pellet in 4 mL gel filtration buffer (20 mM sodium phosphate or potassium phosphate, pH 7.4, 0.1 mM EDTA). Run 5 &#181;L of the resuspended tRNA pool on a 2% w/v agarose gel. 
		NOTE: Good quality tRNA appears as a single band (molecular size between 50-100 bp).</li>
	</ol>
	</li>
	<li>Purification of tRNA(UUU)
	<ol>
		<li>Following standard chromatographic practice12, load the resuspended tRNA(UUU) onto a high-resolution preparative gel filtration column (dimensions: 16 x 600 mm, bed volume: 120 mL, resolution range: 1,000-70,000 Da) previously equilibrated in gel filtration buffer (mobile phase). Set the flow rate to 1 mL/min to elute the samples. 
		NOTE: The tRNA(UUU) peak elutes approximately at 75.5 mL. Typically, a yield of 0.5-1.0 mg/L tRNA of culture can be obtained.</li>
		<li>Analyze the samples at representative elution volumes by electrophoresing 5 - 10 &#181;L of each sample on a 2% w/v agarose gel.</li>
	</ol>
	</li>
</ol>
3. Sample Preparation
<ol>
	<li>Express and purify TcdA according to L&#243;pez-Estepa et al.2. 
	NOTE: Up to 250 &#181;M TcdA can be used for the study.</li>
	<li>Pipette the corresponding amount of TcdA as per Table 1 into properly labeled 1.5 mL centrifuge tubes. Add 1.6 mM tris(2-carboxyethyl)phosphine (TCEP) (final concentration) and gently mix using a pipette. Close the lid and incubate the mixture at room temperature for 5 min.</li>
	<li>Add 10 &#181;M of adenosine triphosphate (ATP) and 50 mM MgCl2 (final concentrations). Mix with a pipette and incubate the mixture for 5 min at room temperature.</li>
	<li>Add purified tRNA(UUU) (section 2.3) as per Table 1, mix with a pipette and incubate at room temperature for 5 min.</li>
	<li>Add phosphate buffered saline (PBS) up to 100 &#181;L final volume. Mix properly and add glycerol to a final concentration of 10% v/v.</li>
	<li>Vortex the sample and briefly spin down the contents of the tube (10,000 x g, 1 min, 4 &#176;C).</li>
</ol>
4. Gel Loading and Electrophoresis Development
<ol>
	<li>Place electrophoresis unit into a tray filled with crushed ice to maintain the chamber at 4 &#176;C during the electrophoresis run.</li>
	<li>Carefully, pipette 5 &#181;L of each sample into the corresponding well. Add a suitable DNA size marker.</li>
	<li>Plug the electrodes into the corresponding slots of the power supply unit. Turn on the power supply unit and set the voltage to 100 V and run the gel for 90 min.</li>
</ol>
5. Gel Staining to Observe tRNA-Protein Interaction
<ol>
	<li>Prepare the staining solution by mixing 50 mL of TBE with 1x of a suitable DNA stain. Avoid exposing the staining solution to sunlight by covering the staining container with aluminum foil. 
	NOTE: DNA stains are usually stored as 10,000 or 20,000x concentrated stocks.</li>
	<li>After 90 min of electrophoresis, carefully remove the gel from the tray and place it in a container with staining solution. Place it on a rocker at low rocking speed at room temperature for 30 min.</li>
	<li>Remove the agarose gel from the staining container, tap it carefully on a piece of cellulose tissue or filter paper to remove excess liquid, and place it directly on a transilluminator. Switch on the UV light to visualize bands containing tRNA. Take a photograph of the gel.</li>
	<li>Protein staining (optional).
	<ol>
		<li>Discard the DNA staining solution safely (according to safety procedures) and substitute it with 50 mL Coomassie Brilliant Blue (CBB) solution. Place the tray on a rocker at room temperature and stain overnight (&#62;12 h). Discard the CBB staining solution and replace it with 50 mL PBS to remove excess dye. Destain for 1 h on a rocker.</li>
	</ol>
	</li>
	<li>Visualize protein-containing tRNA complexes under a light transilluminator and take photographs of the gel to compare with the tRNA UV gel picture.</li>
</ol>

<ol><li>Scott, V., Clark, A. R., Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+gel+retardation+assay%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">The gel retardation assay.</a> Methods Mol Biol. 31, 339-347 (1994).</li>
<li>López-Estepa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+crystal+structure+and+small-angle+X-ray+analysis+of+CsdL%2FTcdA+reveal+a+new+tRNA+binding+motif+in+the+MoeB%2FE1+superfamily%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">The crystal structure and small-angle X-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily.</a> Plos One. 10 (4), e0118606(2015).</li>
<li>Miyauchi, K., Kimura, S., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cyclic+form+of+N6-threonylcarbamoyladenosine+as+a+widely+distributed+tRNA+hypermodification%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">A cyclic form of N6-threonylcarbamoyladenosine as a widely distributed tRNA hypermodification.</a> Nat Chem Biol. 9 (2), 105-111 (2013).</li>
<li>Bolstad, H. M., Botelho, D. J., Wood, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+analysis+of+protein-protein+interactions+within+the+Cysteine+Sulfinate+Desulfinase+Fe-S+cluster+biogenesis+system%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Proteomic analysis of protein-protein interactions within the Cysteine Sulfinate Desulfinase Fe-S cluster biogenesis system.</a> J Proteome Res. 9 (10), 5358-5369 (2010).</li>
<li>Loiseau, L., Ollagnier-de Choudens, S., Lascoux, D., Forest, E., Fontecave, M., Barras, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+heteromeric+CsdA-CsdE+cysteine+desulfurase%2C+assisting+Fe-S+cluster+biogenesis+in+Escherichia+coli%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Analysis of the heteromeric CsdA-CsdE cysteine desulfurase, assisting Fe-S cluster biogenesis in Escherichia coli.</a> J Biol Chem. 280 (29), 26760-26769 (2005).</li>
<li>Fried, M. G., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equilibrium+studies+of+the+cyclic+AMP+receptor+protein-DNA+interaction%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Equilibrium studies of the cyclic AMP receptor protein-DNA interaction.</a> J Mol Biol. 172 (3), 241-262 (1984).</li>
<li>Garner, M. M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions%3A+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9 (13), 3047-3060 (1981).</li>
<li>Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+mobility+shift+assays+for+RNA-protein+complexes%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Electrophoretic mobility shift assays for RNA-protein complexes.</a> Cold Spring Harb Protoc. 2014 (4), 435-440 (2014).</li>
<li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li>
<li>Yakhnin, A. V., Yakhnin, H., Babitzke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gel+mobility+shift+assays+to+detect+protein-RNA+interactions%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Gel mobility shift assays to detect protein-RNA interactions.</a> Methods Mol Biol. 905, 201-211 (2012).</li>
<li>Ream, J. A., Lewis, L. K., Lewis, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+agarose+gel+electrophoretic+mobility+shift+assay+for+quantitating+protein%3A+RNA+interactions%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.</a> Anal Biochem. 511, 36-41 (2016).</li>
<li>Hagel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gel-filtration+chromatography%5BTitle%5D)+AND+%22Curr+Protoc+Mol+Biol%22%5BJournal%5D)">Gel-filtration chromatography.</a> Curr Protoc Mol Biol. , Chapter 10, Unit 10.9 (2001).</li>
</ol>

The authors have nothing to disclose.

The protein-tRNA agarose gel retardation assay described herein can be modified in a number of ways. First, the percentage of agarose in the gel can be reduced to allow the separation and visualization of protein-tRNA complexes significantly larger than the one analyzed here (120 kDa). Second, if the target protein is thermolabile, extra precautions must be taken to ensure that the temperature is kept below the maximum acceptable temperature by moving the electrophoresis apparatus to a cold room or by using ice packs ins...

The gel retardation assay1 (also known as electrophoretic mobility shift assay, EMSA) is an electrophoretic method designed to study and characterize protein-nucleic acid interactions. It allows the analysis of protein-DNA as well as protein-RNA interactions and in particular, interactions between tRNA and tRNA-binding proteins, using either purified components or complex mixtures of proteins (e.g., cell lysates) or nucleic acids (e.g., tRNA pools). We have applied gel retardation assays to the study of the interaction between purified tRNA(UUU) and TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6 (ct6A)2,3. TcdA is also known as CsdL3,4. The tcdA/csdL gene forms a cluster with the csdA and csdE genes5, which encode the cysteine desulfurase and the sulfur acceptor of the cysteine sulfinase desulfinate (CSD) system and is required to sustain TcdA function in vivo3.
The rationale behind the gel retardation assays is that, while free nucleic acid molecules migrate rapidly to the front of acrylamide or agarose gels by virtue of their large negative charge, the electrophoretic mobility of protein complexes of the same nucleic acids is dramatically reduced. The reduction in mobility is visualized as a &#34;shift&#34; in the nucleic acid-protein complexes, which resolve as discrete bands. Positively charged and larger nucleic acid-protein complex show a greater shift or reduction in mobility on a gel.
Since charge neutralization of the complex is a universal phenomenon for protein-nucleic acid complexes, the gel retardation assay can be applied to a wide range of complexes and nucleic acid types. The method is simple, inexpensive, and can be conducted in laboratories with minimum equipment. It requires only small amounts of proteins and tRNA. This is an advantage over alternative biophysical techniques, which usually consume larger quantities of sample.
The gel retardation assay has been widely used for the study of transcription factors. The assay has been used to analyze binding kinetics, strength, and specificity of transcription factors for many different DNA sequences. It was indeed in this field that the EMSA was first developed6,7. Gel retardation assays with RNA8,9,10,11 and tRNA molecules have also been used in ribosome research and to analyze, as we do here, the enzymes that modify tRNA nucleosides post-transcriptionally.
Many different parameters affect the result of a gel retardation assay such as temperature, quality of the protein and tRNA sample, and the strength of the binding. Careful planning and execution of the experiment is crucial for the interpretation of the results of the free nucleic acid and protein-bound species. Here, we used the synthetic gene encoding tRNA(UUU) cloned into an expression vector whose promoter lacks the Shine-Dalgarno ribosome binding site, leading to the synthesis of large amounts of the tRNA2. This detailed protocol is intended to provide a clear guide for performing tRNA gel retardation experiments while avoiding common mistakes.

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21(DE3)</td><td>Novagen</td><td>70235</td><td>DE3 lysogen contains T7 polymerase upon IPTG induction.</td></tr><tr><td>pET23d</td><td>Novagen</td><td>69748-3</td><td>pET23d confers resistance to ampicillin; T7 promoter.</td></tr><tr><td>LB Broth, Low Salt (Lennox L Broth), Granulated</td><td>Melford</td><td>GL1703</td><td>Conventional LB Broth, High Salt, can also be used.</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>10419313</td><td></td></tr><tr><td>Incubator Shaker (Thermostated)</td><td>NBS</td><td>Excella E24&amp;nbsp;</td><td>Any shaker incubator with temperature control can be used.</td></tr><tr><td>Isopropyl-beta-D-thiogalactopyranoside (IPTG), &gt;=99%</td><td>Acros Organics</td><td>BP1755</td><td></td></tr><tr><td>Sodium acetate, anhydrous, &gt;99%</td><td>Melford</td><td>B4017</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA), Disodium salt, &gt;99%</td><td>Acros Organics</td><td>AC327205000</td><td>Harmful if inhaled.</td></tr><tr><td>Centrifuge (refrigerated)</td><td>Eppendorf</td><td>5430</td><td></td></tr><tr><td>Centrifuge (refrigerated) rotor</td><td>Eppendorf</td><td>5430/5430P</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>RC5C</td><td></td></tr><tr><td>Centrifuge rotor</td><td>Sorvall</td><td>SLA-3000</td><td></td></tr><tr><td>Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 &amp;plusmn; 0.2</td><td>Sigma-Aldrich</td><td>P4682</td><td>Acute toxicity (oral, dermal, inhalation), corrosive</td></tr><tr><td>Ethanol absolute for analysis, 100% v/v</td><td>Merck Millipore</td><td>100983</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, &gt;=99%</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>Potassium dihydrogen phosphate, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>48,730,250</td><td></td></tr><tr><td>HiLoad 16/60 Superdex 75&amp;nbsp;</td><td>GE Healthcare</td><td>28989333</td><td></td></tr><tr><td>Agarose</td><td>Melford</td><td>MB1200</td><td></td></tr><tr><td>Tris [Tris(hydroxymethyl) aminomethane HCl]</td><td>Melford</td><td>T1513</td><td></td></tr><tr><td>Boric Acid</td><td>Melford</td><td>B0503</td><td></td></tr><tr><td>Microwave</td><td>Haier</td><td>HDA-2070M</td><td></td></tr><tr><td>TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]</td><td>Sigma-Aldrich</td><td>C4706</td><td>Corrosive to metals and skin.</td></tr><tr><td>ATP [Adenosine 5&amp;prime;-triphosphate disodium salt hydrate], Grade I, &gt;=99%</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;), anhydrous, &gt;=98%</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Sodium chloride (NaCl), &gt;99%</td><td>Fisher Bioreagents</td><td>BP358</td><td></td></tr><tr><td>Potassium chloride (KCl), &gt;99%</td><td>Melford</td><td>P0515</td><td></td></tr><tr><td>NZYDNA Ladder VI</td><td>NZYtech</td><td>MB8901</td><td></td></tr><tr><td>Power supply</td><td>Consort</td><td>EV261</td><td></td></tr><tr><td>Electrophoresis unit</td><td>Real Laboratory</td><td>RELSMINI10</td><td></td></tr><tr><td>Real Safe nucleic acid staining</td><td>Real Laboratory</td><td>RBMSAFE</td><td>20,000X stock</td></tr><tr><td>UV Transilluminator (G:Box/EF)</td><td>Syngene</td><td>2216498</td><td></td></tr><tr><td>Coomassie Brilliant Blue G</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Rocker (Gyro-Rocker)</td><td>Stuart</td><td>SSL3</td><td></td></tr><tr><td>Mixer Vortex&amp;nbsp;</td><td>Fisher brand</td><td>13214789</td><td></td></tr></tbody></table>

protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Large amounts of tRNA(UUU) can be obtained by expressing the tRNA gene in E. coli under the control of a strong inducible T7 promoter. The expressed tRNA(UUU) accumulates in the cytoplasm and is enriched over the pool of naturally abundant tRNAs. A two-step purification process consisting of a capture/extraction step and a gel filtration/polishing step was utilized to obtain EMSA-grade tRNA. The capture/extraction step uses pH 4.3 phenol to achieve the simultaneous precipitation ...

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

The overall goal of this procedure is to demonstrate an RNA gel retardation assay designed for the discovery and characterization of RNA-binding proteins. Application of this assay is used to investigate the interaction between TcdA and transfer RNA. The agarose gel retardation method can provide answers to key questions in the field of RNA biology.For example, whether a certain protein interacts with RNA, whether the interaction is sequences-specific, and the strength of the interaction. The main advantages of this technique are that it uses various small amounts of protein and RNA samples, and yields very informative results about the protein-RNA interactions quite quickly. Though this method can provide insight into protein-tRNA complexes, it can also be applied to many other systems such as ribosomal RNA, messenger RNA, and any other kinds of RNA molecule.In the morning, retrieve a vial of frozen E coli BL21 DE3 transformed with the expression vector pET-23D EC triple-U. Streak a low salt luria broth or LB plate supplemented with 100 micrograms per milliliter ampicillin. Invert the plate and place it into a 37 degrees Celsius incubator until colonies appear in the late afternoon.Inoculate one well-grown single colony in five milliliters of LB plus 100 micrograms per milliliter of ampicillin and grow overnight. The next morning, pellet the cells. Following centrifugation, replace the medium with five milliliters of fresh LB plus 100 micrograms per milliliter of ampicillin and re-suspend.Use this suspension to inoculate 200 milliliter of LB plus 100 micrograms per milliliter of ampicillin. Grow the cells for five hours at 220 RPM and 37 degrees Celsius. When the optical density of the culture at 590 nanometers has reached 0.6 to 0.8, atdd1 millimolar IPTG to the culture to trigger the expression of the tRNA triple-U gene.After incubating the cells at 37 degrees Celsius for three hours, harvest the cells by centrifuging the culture at 6500 times g for 10 minutes at four degrees Celsius. To perform lysis, first re-suspend the cell pellet in five milliliters of lysis buffer. Add one volume of acid phenol to the re-suspended cell pellet in the fume hood and mix for one minute by inversion to lyse the cells.Then, centrifuge at 10 thousand times g for 10 minutes at 4 degrees Celsius. Collect the upper aqueous phase containing the soluble tRNA triple-U using a pipette. Take care not to aspirate the organic bottom phase.Precipitate the tRNA by thoroughly mixing the soluble phase with 2.5 volumes of 100%volume per volume ethanol by inversion for one hour at four degrees Celsius. Then, centrifuge at 15 thousand times g for 30 minutes at four degrees Celsius. Decant the supernatant carefully, then resuspend the tRNA-rich pellet in four milliliters of gel filtration buffer.Following standard chromatographic practice, load the re-suspended tRNA triple-U onto a high-resolution preparative gel filtration column previously equilibrated in gel filtration buffer. Set the flow rate to 1 milliliter per minute to elute the samples. To begin sample preparation, pipette the corresponding amount of TcdA as indicated in the text protocol into properly labeled 1.5 milliliter centrifuge tubes.Add a final concentration of 1.6 millimolar TCEP and gently mix using a pipette. Close the lid and incubate the mixture at room temperature for five minutes. Following incubation, add adenosine triphosphate and magnesium chloride.Mix with a pipette before incubating the mixture for five minutes at room temperature. Next, add purified tRNA triple-U according to the text protocol and mix the sample with a pipette. After incubating the sample at room temperature for five minutes, add phosphate buffered saline up to a 100 microliter final volume.Mix properly and add glycerol to a final concentration of 10%Vortex the sample before briefly spinning down the contents of the tube. Prepare the agarose gel as described in the text protocol. Place the electrophoresis unit into a tray filled with crushed ice to maintain the chamber at four degrees Celsius during the electrophoresis run.Carefully pipette five microliters of each sample into the corresponding well. Also add a suitable DNA size marker. Then, plug the electrodes into the corresponding slots of the power supply unit.After turning on the power supply unit, set the voltage to 100 volts and run the gel for 90 minutes. Next, prepare the staining solution by mixing 50 milliliters of TBE with 1X of a suitable DNA stain. After 90 minutes of electrophoresis, carefully remove the gel from the tray and place it in a container with staining solution.Place the container on a rocker at low rocking speed and rock at room temperature for 30 minutes. Remove the agarose gel from the staining container and tap it carefully on a piece of cellulose paper to remove excess liquid. Then, place the gel directly on a transilluminator.Switch on the UV light to visualize bands containing tRNA. Finally, take a photograph of the gel. Shown here are representative results of tRNA purification for gel retardation assays.In the chromatogram the absorbance of the elution fractions at 254 nanometers is recorded. The tRNA pool, enriched in over-expressed tRNA triple-U elutes at the highest peak. The quality and quantity of the tRNA pool is examined by agarose gel electrophoresis.The RNA gel retardation assay reveals that TcdA forms a stable complex with tRNA triple-U in tRNA concentration-dependent manner, which results in a protein-tRNA band that migrates more slowly than the free tRNA at the front of the gel. Once mastered, this technique can be done in less than two hours. While attempting this protocol, it's important to remember to follow strict laboratory techniques to ensure the chemical stability of tRNA.After watching this video, you should have a good understanding how to use agarose gel retardation assay to investigate the interaction of your protein of choice with tRNA, as in the case of TcdA or with any other type of RNA.

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Research

JoVE Journal

Biochemistry

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In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

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An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

CD Spectroscopy to Study DNA-Protein Interactions

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

Learn agarose gel retardation assays to analyze TcdA-tRNA interactions and characterize RNA-binding proteins. This method enables rapid, sequence-specific protein-RNA binding studies using minimal sample amounts.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N⁶-threonylcarbamoyladenosine TcdA Function

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