This work acknowledges a Faculty Early Career Grant from the University of Newcastle (CM).

Caution: Experiments involve the use of the radioactive isotope 32P and no work should be undertaken until all appropriate safety conditions have been met. Generally personnel are required to attend a safety course and undergo supervised practice prior to experiments using radioactive reagents. Please wear personal protection equipment (thermoluminescent dosimeter, safety glasses, gloves, radiation room lab coats, full length pants, closed-toe shoes) when performing the reaction.
1. Preparation of Assay Materials
<ol>
	<li>Obtain the Proteins Required for Transcription assay.
	<ol>
		<li>For the experiment described here, overproduce and purify E. coli RNAP in the laboratory as described11, or obtain from a commercial source (Materials List). 
		Note: Both provide similar results. Other methods for purification of native or affinity labeled RNAP can also be used12,13. Sigma/NusA factors can be obtained as described in previous work14,15.</li>
	</ol>
	</li>
	<li>Preparation of Diethyl Pyrocarbonate (DEPC) Treated Water 
	Note: Alternatively, RNase-free water can be obtained from commercial sources (Materials List).
	<ol>
		<li>Add 1 ml diethyl pyrocarbonate (DEPC) to 1 L of purified water (0.1% v/v) and mix overnight.</li>
		<li>Autoclave DEPC water and tips twice.</li>
	</ol>
	</li>
	<li>DNA Template Preparation
	<ol>
		<li>Purify plasmid pSG115416 and/or pIA22617 using a plasmid purification kit (Materials List) according to manufacturer&#39;s instructions.</li>
		<li>To amplify the template for run-off transcription assays (a 360 bp fragment encompassing the Pxyl promoter region), assemble a PCR reaction on ice including 25 &#956;l 2x PCR mix (Materials List), 2 &#956;l each of the primers (5 &#181;M): 5&#8242;-CAAAGCCTGTCGGAATTGG-3&#8242; (forward) and 5&#8242;-CCCATTAACATCACCATC-3&#8242; (reverse), 1 &#956;l pSG1154 plasmid preparation (250 &#181;M) and 20 &#956;l purified water).
		<ol>
			<li>Or alternatively, for single round transcription assays, amplify a 447 bp fragment encompassing the &#955; PR promoter region from pIA22617. Set up a PCR reaction on ice using 25 &#956;l 2x PCR mix, 2 &#956;l each of the primers (5 &#181;M): 5&#8242;-GTGCTCATACGTTAAATCT-3&#8242; (forward) and 5&#8242;-CAGTTCCCTACTCTCGCATG-3&#8242; (reverse), 1 &#956;l pIA22617 plasmid preparation (250 &#181;M) and 20 &#956;l purified water). 
			Note: This template lacks cytosine until the +26 nt position, which is the 26th nucleotide from the transcription start site. Therefore, transcription using this template will allow initiation, promoter clearance and stalling at the +26 nt to form a stable transcription elongation complex (EC) when only ATP, UTP and GTP are present in the reaction.</li>
		</ol>
		</li>
		<li>Set up a PCR reaction with the following conditions: 95 &#176;C for 30 sec, 50 &#176;C for 30 sec, 72 &#176;C for 30 sec, repeating for 30 cycles.</li>
		<li>Purify the product using a PCR clean-up kit (Materials List) according to manufacturer&#39;s instructions and elute in 100 &#956;l transcription buffer for the transcription assay.</li>
		<li>Determine the template DNA concentration using a fluorospectrometer (Materials List).</li>
	</ol>
	</li>
	<li>NTP Preparation 
	Note: NTPs can be obtained from different commercial sources (Materials List).
	<ol>
		<li>Dissolve NTPs (&#8805;99% pure) in DEPC-treated water to make 1 M stock, aliquot 1 ml of the solution into 1.5 ml tubes (50 &#956;l in each tube) and store at -20 &#176;C.</li>
		<li>Order &#945;-32P UTP (3,000 Ci/mmol) prior to the experiment. 32P has a short half-life of 14.29 days. 
		Note: Using alpha-labeled UTP avoids comparing the intensity of bands with different length.</li>
	</ol>
	</li>
	<li>RNA Ladder Preparation
	<ol>
		<li>Assemble the following reaction at room temperature: 0.5 &#956;g RNA Template Mix (Materials List), 80 mM HEPES buffer pH7.5, 12 mM MgCl2, 10 mM NaCl, 10 mM DTT, 2 mM ATP/CTP/GTP, 0.5 mM UTP, 2 &#956;l &#945;-32P UTP, 0.5 &#956;l T7 RNAP (Materials List), DEPC-treated water to 20 &#956;l.</li>
		<li>Allow reaction to proceed at 37 &#176;C for 1 hr. Use immediately or store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of RNA Sequencing Gel
<ol>
	<li>Gel Preparation
	<ol>
		<li>Clean the plates from a sequencing gel apparatus (Materials List) twice with 70% ethanol and purified water, and assemble the cell with a comb and two spacers. 
		Note: One plate can be coated with siliconizing reagent (Materials List) to facilitate gel removal in Step 4.2.</li>
		<li>Dissolve 33.6 g urea in 22 ml water and 16.4 ml of 5x Tris/Borate/EDTA (TBE) buffer (54 g tris(hydroxymethyl)aminomethane; 27.5 g boric acid; 10 ml of 1 M ethylenediaminetetraacetic acid (EDTA), pH 7.4 in 1 L water) using a magnetic stirrer.</li>
		<li>Add 16 ml of 40% Acrylamide/bis-acrylamide solution (19:1) to the urea solution and filter through a 0.45 &#181;m filter.</li>
		<li>Add 514 &#181;l 10% (w/v) freshly prepared ammonium persulfate in water followed by 51.4 &#181;l N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine (TEMED) to the filtered solution.</li>
		<li>Gently invert the gel solution to avoid excessive aeration and inject immediately and smoothly into the horizontally positioned pre-packed sequencing cell from the bottom injection hole. Take care to avoid making bubbles during injection.</li>
		<li>Allow 1-1.5 hr until the gel is completely set before use in a transcription assay. The gel can be stored at room temperature for two or three days when wrapped with water-wet tissue on the top of the sequencing cell.</li>
	</ol>
	</li>
</ol>
3. Transcription Reaction
<ol>
	<li>Transcription Elongation Pausing Assay
	<ol>
		<li>Formation of a transcription elongation complex
		<ol>
			<li>Add 1 &#181;l of RNAP core enzyme (1 &#181;M stock in transcription buffer) to a 1.5 ml tube.</li>
			<li>Add 1 &#181;l of purified sigma-70 factor (&#963;70; 3 &#181;M stock in transcription buffer) to the RNAP and incubate on ice for 10 min to form RNAP holoenzyme.</li>
			<li>Add 3 &#181;l 500 nM purified template DNA (&#955; PR promoter from the pIA226 PCR product) in transcription buffer to the RNAP holoenzyme and incubate at 37 &#186;C for 5 min to form the open complex.</li>
			<li>Prepare a mixture of reaction components in another 1.5 ml tube using 5 &#181;l of 10x transcription buffer (Materials List: 400 mM Tris-HCl, 1.6 M KCl, 100 mM MgCl2, 10 mM DTT, 50% glycerol, pH 7.9 in DEPC treated water), 2 &#181;l of 250 &#181;M each GTP and UTP, 1 &#181;l of 1 mM ATP, and 1 &#181;l of &#945;-32P UTP (3,000 Ci/mmol). Add DEPC treated water to 41 &#181;l. 
			Note: RNase inhibitor (Materials List) can be used to minimize the RNase contamination.</li>
			<li>Transfer the mixture solution to the tube containing the open complex and incubate at 37 &#186;C for 15 min to form the transcription elongation complex stopped at +26 nt.</li>
			<li>Move the reaction to room temperature (~21 &#176;C) and add 1 &#181;l of 2.5 mg/ml rifampicin to a final concentration of 50 &#181;g/ml.</li>
			<li>Add 1 &#181;l of 0.1 mM purified NusA to the reaction mixture and incubate for 5 min at room temperature.</li>
			<li>Add 2 &#181;l of 250 &#181;M CTP into the reaction mixture to make a final volume of 50 &#181;l and measure the reaction time using a timer.</li>
			<li>Transfer 5 &#181;l of the reaction solution into 2 &#181;l of RNA gel loading buffer (95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol) at 30, 60, 120, 240, 480, and 1,200 sec to quench the reaction.</li>
			<li>Dispose the 32P UTP tips and tubes in a radioactive waste container.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transcription Initiation Assay
	<ol>
		<li>Formation of the Open Complex&#160;
		<ol>
			<li>Dilute purified &#963;70 to 0.5 &#181;M.</li>
			<li>Repeat Steps 3.1.1.1 to 3.1.1.3 using 0.5 &#181;M (instead of 3 &#181;M) purified &#963;70, and the DNA template (the Pxyl promoter region from pSG1154 PCR product).</li>
		</ol>
		</li>
		<li>Inhibition of transcription initiation assay
		<ol>
			<li>Add the inhibitor designed to prevent RNAP holoenzyme formation to RNAP core enzyme prior to addition of &#963;70 (after step 3.1.1.1), followed by Steps 3.1.1.2 and 3.1.1.3 to examine whether the inhibitor is able to disrupt holoenzyme formation.</li>
			<li>Alternatively, after Step 3.1.1.2, add the inhibitor to examine whether it is able to competitively inhibit &#963;70 binding to RNAP.</li>
			<li>Use the same amount of DMSO, the solvent of the inhibitor, instead of the inhibitor stock in control reactions.</li>
		</ol>
		</li>
		<li>Completion of the reaction
		<ol>
			<li>Dissolve heparin in water to a stock concentration of 1 mg/ml and stock at -20 &#176;C. 
			Note: Heparin is a competitor of DNA that can sequester RNAP that has not formed an open complex. Once the open complex is formed, heparin cannot enter the DNA binding site of RNAP and inhibit transcription from pre-formed open complex. Therefore, heparin is only required for single-round assays; it is not needed for multi-round assays.</li>
			<li>Prepare the reaction mixture using 5 &#181;l of 10x transcription buffer (Materials List), 1 &#181;l of 1 mg/ml heparin solution, 1 &#181;l of 10 mM each ATP, GTP, and CTP, 2.5 mM UTP, and 1 &#181;l of &#945;-32P UTP (3,000 Ci/mmol). Add DEPC treated water to 45 &#181;l.</li>
			<li>Mix the reaction mixture with the transcription open complex, pulse spin and incubate at 37 &#176;C for 20 min.</li>
			<li>Transfer 10 &#181;l of the reaction solution into 5 &#181;l of RNA gel loading buffer (95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol) to quench the reaction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. RNA Gel Running and Development
<ol>
	<li>Running the Denaturing Polyacrylamide Gel
	<ol>
		<li>Run the gel at 50 &#186;C and 50 W in 1x TBE buffer for approximately 1.5 hr until the temperature of the gel and the buffer is stable at 50 &#186;C.</li>
		<li>Heat samples at 90 &#186;C for 30 sec and move them onto ice prior loading onto the gel.</li>
		<li>Load samples into the wells on the top of the gel alongside one lane containing 2 &#181;l RNA ladder (1:100 dilution).</li>
		<li>Run the gel at 50 W and 50 &#186;C in 1x TBE buffer until the bromophenol blue dye reaches ~2/3 down the gel.</li>
	</ol>
	</li>
	<li>Gel Treatment
	<ol>
		<li>Open slowly and carefully separate the upper plate from the sequencing cell. Take great care to avoid breaking the gel.</li>
		<li>Cut chromatography paper (Materials List) to match the gel size from the top to the bromophenol blue dye. Use a Geiger counter to confirm the approximate position of radioactively labelled RNA transcripts.</li>
		<li>Carefully cover the gel with the filter paper, and press firmly till the gel adheres to the filter paper. Cut off and discard the bottom of the gel to radioactive waste as the unincorporated NTPs run to the bottom of the gel, which can be very hot.</li>
		<li>Place a similar sized piece of filter paper on the drying bed of a vacuum-drying machine, and carefully lay the gel-coated paper onto it with the gel side up.</li>
		<li>Cover the gel with a plastic wrap and dry at 60 &#176;C for 1.5 hr.</li>
		<li>Wrap the other side of the gel coated filter paper with plastic wrap, and place on a photostimulable phosphor plate in the imaging case, with the appropriate exposure time, which can vary from 1 hr to overnight depending on the reaction efficiency.</li>
		<li>Image the phosphor plate using an imager scanner according to manufacturer&#39;s instructions. Determine the relative band intensity using ImageJ software according to the software manual.</li>
		<li>Use a Geiger counter to check the radioactive contamination in the work area, wipe the suspected items and area with tissue and a cleaning agent and dispose the tissue in the disposal container.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In all organisms, transcription is a tightly regulated process. In vitro transcription assays have been developed to provide a platform for testing the effects of transcription factors, small molecules and transcription inhibitors. In this method paper, an assay for general bacterial transcription was described. Transcription assays combined with sequencing gel electrophoresis of transcripts are very important for mechanistic studies as they allow visualization of all the transcription products along a timeline....

Transcription is the process in which RNA is synthesized from a specific DNA template. In eukaryotic cells there are three distinct RNAPs: RNAP I transcribes rRNA precursors, RNAP II is responsible for the synthesis of mRNA and certain small nuclear RNAs, and the synthesis of 5S rRNA and tRNA is performed by RNAP III. In bacteria, there is only one RNAP responsible for the transcription of all classes of RNA. There are three stages of transcription: initiation, elongation and termination. Transcription is one of the most highly regulated processes in the cell. Each stage in the transcription cycle represents a checkpoint for the regulation of gene expression1. For initiation, RNAP has to associate with a sigma factor to form holoenzyme, which is required to direct the enzyme to specific sites called promoters2 to form an open promoter complex. Subsequently, a large suite of transcription factors are responsible for the regulation of RNAP activities during the elongation and termination phases. The transcription factor examined here is the highly conserved and essential protein, NusA. It is involved in regulating transcription pausing and termination, as well as anti-termination during rRNA synthesis3-5.
In vitro transcription assays have been developed as powerful tools to study the complex regulatory steps during transcription6. In general, a linear fragment of DNA including a promoter region is required as the template for transcription. The DNA template is usually generated by PCR or by linearizing a plasmid. Purified proteins and NTPs (including one radiolabeled NTP for detection purposes) are then added and product analyzed following the required period of incubation. Using appropriate templates and reaction conditions, all stages of transcription have been examined using this approach which has enabled detailed molecular characterization of transcription over the last half century7. In combination with information on the 3-dimensional structure of RNAP it has also been possible to probe the molecular mechanism of transcription inhibition by antibiotics and antibiotic leads, and use this information in the development of new, improved drugs8-10.
In this work we provide examples of how transcription assays can be used to determine the mechanism of regulation by transcription elongation/termination factor NusA, and how the mechanism of action of a novel transcription initiation inhibitor lead can be determined.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Obtain the proteins required for transcription assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAP</td>
			<td>Epicentre</td>
			<td>S90250</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of DEPC-treated water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl pyrocarbonate (DEPC)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5758</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion Nuclease-Free Water</td>
			<td>ThermoFisher</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA template preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard&#160;Plus&#160;SV Minipreps DNA Purification System</td>
			<td>Promega</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>ACCUZYME Mix</td>
			<td>Bioline</td>
			<td>BIO-25028</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 3300 fluorospectrometer</td>
			<td>Thermo Scientific</td>
			<td>ND-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>NTP Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A6559</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>U1006</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G3776</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C9274</td>
			<td></td>
		</tr>
		<tr>
			<td>High Purity rNTPs</td>
			<td>GE Healthcare</td>
			<td>27-2025-01</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-32P UTP&#160;</td>
			<td>PerkinElmer&#160;&#160;</td>
			<td>BLU007C001MC</td>
			<td>Radioactive compound</td>
		</tr>
		<tr>
			<td>RNA ladder preparation&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Novagen Perfect RNA Marker Template Mix 0.1&#8211;1 kb</td>
			<td>Millipore</td>
			<td>69003</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNAP</td>
			<td>Promega</td>
			<td>P2075</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequi-Gen GT nucleic acid sequencing cell&#160;</td>
			<td>Bio-Rad&#160;&#160;</td>
			<td>165-3804</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>SL2</td>
			<td></td>
		</tr>
		<tr>
			<td>urea&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>U6504</td>
			<td></td>
		</tr>
		<tr>
			<td>tris(hydroxymethyl)aminomethane&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr>
			<td>boric acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>B7901</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/bis-acrylamide&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9926</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium persulfate&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#697;&#8242;,N&#697;&#8242;-Tetramethylethylenediamine (TEMED)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N",N"-Tetramethylethylenediamine (TEMED) </td>
			<td>Sigma-Aldrich </td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>rifampicin&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene cyanol&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>heparin&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>84020</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman&#160;3MM Chr Chromatography Paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Radioactive decontaminant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Decon 90</td>
			<td>decon</td>
			<td>decon90</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon Trio+ imager</td>
			<td>GE Healthcare Life Sciences</td>
			<td>63-0055-89</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro transcription assays and their application in drug discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

Transcription efficiency can be determined by measuring the level of radiation in the bands at different time points. The pausing assay to test the function of NusA factor enabled visualization of the pause, termination, and run-off products (Figure 1A). In the presence of the N-terminal domain of NusA (NusA NTD; amino acid residues 1-137), appearance of the RNA products was significantly delayed compared to the control experiment lacking NusA. Deletion of amino acid resi...

Watch this Scientific Journal Video about In Vitro Transcription Assays and Their Application in Drug Discovery at JoVE.com

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

in-vitro-transcription-assays-and-their-application-in-drug-discovery

Research

JoVE Journal

Genetics

15.0K Views.  University of Newcastle. In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

Video: In Vitro Transcription Assays and Their Application in Drug Discovery

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...