Name: protein-trna agarose gel retardation assays for the analysis of the n6-threonylcarbamoyladenosine tcda function
Uploaded: 2017-06-21T12:30:00
Description: Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

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...

<ol>
	<li>Scott, V., Clark, A. R., Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+retardation+assay.">The gel retardation assay.</a> Methods Mol Biol. 31, 339-347 (1994).</li><li>L&#243;pez-Estepa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+and+small-angle+X-ray+analysis+of+CsdL/TcdA+reveal+a+new+tRNA+binding+motif+in+the+MoeB/E1+superfamily.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyclic+form+of+N6-threonylcarbamoyladenosine+as+a+widely+distributed+tRNA+hypermodification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+protein-protein+interactions+within+the+Cysteine+Sulfinate+Desulfinase+Fe-S+cluster+biogenesis+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+heteromeric+CsdA-CsdE+cysteine+desulfurase,+assisting+Fe-S+cluster+biogenesis+in+Escherichia+coli.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+studies+of+the+cyclic+AMP+receptor+protein-DNA+interaction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assays+for+RNA-protein+complexes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+mobility+shift+assays+to+detect+protein-RNA+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+agarose+gel+electrophoretic+mobility+shift+assay+for+quantitating+protein:+RNA+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-filtration+chromatography.">Gel-filtration chromatography.</a> Curr Protoc Mol Biol. , (2001).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">E. coli BL21(DE3)</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:119px;">70235</td>
			<td style="width:367px;">DE3 lysogen contains T7 polymerase upon IPTG induction.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:433px;">pET23d</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:119px;">69748-3</td>
			<td style="width:367px;">pET23d confers resistance to ampicillin; T7 promoter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">LB Broth, Low Salt (Lennox L Broth), Granulated</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">GL1703</td>
			<td style="width:367px;">Conventional LB Broth, High Salt, can also be used.</td>
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			<td style="width:135px;">NBS</td>
			<td style="width:119px;">Excella E24&#160;</td>
			<td style="width:367px;">Any shaker incubator with temperature control can be used.</td>
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			<td height="21" style="height:21px;width:433px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG), &#62;=99%</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:119px;">BP1755</td>
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			<td height="21" style="height:21px;width:433px;">Sodium acetate, anhydrous, &#62;99%</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">B4017</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ethylenediaminetetraacetic acid (EDTA), Disodium salt, &#62;99%</td>
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			<td style="width:119px;">AC327205000</td>
			<td style="width:367px;">Harmful if inhaled.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge (refrigerated)</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:119px;">5430</td>
			<td style="width:367px;"></td>
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			<td height="21" style="height:21px;width:433px;">Centrifuge (refrigerated) rotor</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:119px;">5430/5430P</td>
			<td style="width:367px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Centrifuge</td>
			<td style="width:135px;">Sorvall</td>
			<td style="width:119px;">RC5C</td>
			<td style="width:367px;"></td>
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			<td height="21" style="height:21px;width:433px;">Centrifuge rotor</td>
			<td style="width:135px;">Sorvall</td>
			<td style="width:119px;">SLA-3000</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 &#177; 0.2</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4682</td>
			<td style="width:367px;">Acute toxicity (oral, dermal, inhalation), corrosive</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Ethanol absolute for analysis, 100% v/v</td>
			<td style="width:135px;">Merck Millipore</td>
			<td style="width:119px;">100983</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium phosphate dibasic dihydrate, Na2HPO4, &#62;=99%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">71643</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Potassium dihydrogen phosphate, KH2PO4</td>
			<td style="width:135px;">Merck</td>
			<td style="width:119px;">48,730,250</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">HiLoad 16/60 Superdex 75&#160;</td>
			<td style="width:135px;">GE Healthcare</td>
			<td style="width:119px;">28989333</td>
			<td style="width:367px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Agarose</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">MB1200</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Tris [Tris(hydroxymethyl) aminomethane HCl]</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">T1513</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Boric Acid</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">B0503</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Microwave</td>
			<td style="width:135px;">Haier</td>
			<td style="width:119px;">HDA-2070M</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">TCEP [Tris(2-carboxyethyl)phosphine hydrochloride]</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4706</td>
			<td style="width:367px;">Corrosive to metals and skin.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:433px;">ATP [Adenosine 5&#8242;-triphosphate disodium salt hydrate], Grade I, &#62;=99%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2383</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:433px;">Magnesium chloride (MgCl2), anhydrous, &#62;=98%</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Sodium chloride (NaCl), &#62;99%</td>
			<td style="width:135px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP358</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Potassium chloride (KCl), &#62;99%</td>
			<td style="width:135px;">Melford</td>
			<td style="width:119px;">P0515</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">NZYDNA Ladder VI</td>
			<td style="width:135px;">NZYtech</td>
			<td style="width:119px;">MB8901</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Power supply</td>
			<td style="width:135px;">Consort</td>
			<td style="width:119px;">EV261</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Electrophoresis unit</td>
			<td style="width:135px;">Real Laboratory</td>
			<td style="width:119px;">RELSMINI10</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Real Safe nucleic acid staining</td>
			<td style="width:135px;">Real Laboratory</td>
			<td style="width:119px;">RBMSAFE</td>
			<td style="width:367px;">20,000X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">UV Transilluminator (G:Box/EF)</td>
			<td style="width:135px;">Syngene</td>
			<td style="width:119px;">2216498</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Coomassie Brilliant Blue G</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0770</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Rocker (Gyro-Rocker)</td>
			<td style="width:135px;">Stuart</td>
			<td style="width:119px;">SSL3</td>
			<td style="width:367px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Mixer Vortex&#160;</td>
			<td style="width:135px;">Fisher brand</td>
			<td style="width:119px;">13214789</td>
			<td style="width:367px;"></td>
		</tr>
	</tbody>
</table>

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 ...

Watch this Scientific Journal Video about Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function at JoVE.com

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

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.

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 ...

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