We thank S. Shuman for providing the mammalian capping enzyme cDNA. This work was supported in part by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.&#160;&#160;Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1434.

1. Assembly of Artificial Elongation Complexes and Pol II Walking
<ol>
	<li>Immobilize 1 nmol of non-template DNA oligo containing a 3&#8217; biotin molecule on magnetic beads. 
	NOTE: The following steps can be done in advance to prepare for future experiments. All oligo sequences used in this protocol are provided in Table 1. RNA oligos are synthesized with 5&#8217;-triphosphate modifications.
	<ol>
		<li>Add 200 &#181;L of magnetic beads (10 mg/mL) to a low-protein binding 1.5 mL tube, and then place on a magnetic rack for 2 min.</li>
		<li>While the tube is on the magnetic rack, remove the liquid from the tube without disturbing the beads. To wash the beads, remove the tube from the rack, add 1 mL of 5&#8201;mM Tris-HCl pH 7.5, 0.5&#8201;mM EDTA, 1&#8201;M NaCl, return the tube to the rack, and remove the wash solution after 2 min.</li>
		<li>Repeat washes 2 more times using 200 &#181;L of the same buffer.</li>
		<li>Resuspend magnetic beads in 400 &#181;L of 10&#8201;mM Tris-HCl pH 7.5, 1&#8201;mM EDTA, 2&#8201;M NaCl, buffer. Then mix with 380 &#181;L of H2O and 20 &#181;L of 10 &#181;M non-template biotinylated DNA oligo. Place the tube on a nutator and incubate for 30 min at room temperature.</li>
		<li>Wash immobilized non-template DNA oligo 3 times with 200 &#181;L of 5&#8201;mM Tris-HCl pH 7.5, 0.5&#8201;mM EDTA, 1&#8201;M NaCl, and then 3 times with 200 &#181;L of 20&#8201;mM HEPES-NaOH pH 7.9, 20% glycerol, 100&#8201;mM KCl, 1&#8201;mM EDTA, 0.5&#8201;mg/mL bovine serum albumin.</li>
		<li>Leave tube overnight at 4 &#176;C to finish blocking beads with BSA.</li>
		<li>The next day, place the tube in the magnetic rack, remove and discard the liquid, and&#160;resuspend beads&#160;in 200 &#181;L of 20&#8201;mM HEPES-NaOH pH 7.9, 20% glycerol, 100&#8201;mM KCl, 1&#8201;mM EDTA, 0.5&#8201;mg/mL bovine serum albumin and transfer to a new tube. For this DNA template, final concentration is approximately 5 &#181;M. 
		NOTE: Incubation time and oligo concentration need to be empirically determined for each biotinylated DNA oligo. This template should provide enough for 200 reactions, and last at least 6 months when stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Anneal RNA and DNA template strand oligonucleotide oligos in a 2:1 molar ratio (20 and 10 pmol, respectively) to obtain the DNA:RNA duplex.
	<ol>
		<li>Set up a 10 &#181;L annealing mix as described in Table 2. 
		NOTE: 10 &#181;L of annealing mix are sufficient for 10 reactions. The annealing mix can be scaled up as needed if more assays are to be performed.</li>
		<li>Perform the annealing reactions in a thermal cycler using the following program: 5 min at 45 &#176;C, followed by 12 cycles of 2 min each, starting at 43 &#176;C and decreasing the temperature 2 &#176;C per cycle. Idle at 4 &#176;C. 
		NOTE: This is a flexible timepoint. Annealing is finished within ~30 min but can be left at 4 &#176;C for a longer period. In the lab, annealing mixtures have been left sitting for up to 4 h without observing any decrease in reaction efficiency.</li>
		<li>While annealing RNA to the template strand DNA, prepare buffers for later steps of the protocol. The ingredients needed to prepare each buffer for a single reaction with each buffer are listed in Tables 2 to 8. Scale up recipes by a factor of [Y(X+1) + 1] for Wash and (X+1) for all other buffers. X = number of reactions to be prepared; Y = number of washing steps needed (minimum 3). 
		NOTE: Prepare all buffers fresh the day of the experiment. Do not place tubes on ice after PVA has been added to buffers. Add Pol II or nucleotides to buffers right before using.</li>
	</ol>
	</li>
	<li>Load purified RNA polymerase II onto the DNA:RNA hybrid.
	<ol>
		<li>Mix 1 pmol (1 &#181;L) of DNA:RNA duplex with 13 &#181;L of Pol II buffer (from Table 3).</li>
		<li>Add ~0.02 units of purified RNA Pol II (1 &#181;L) to the mixture from step 1.3.1 and mix by gently pipetting up and down and stirring with pipet tip, without introducing bubbles. Incubate for 10 min at 30 &#176;C. 
		NOTE: Volumes given from here on are for a single reaction; scale up as needed for the number of reactions in the experiment. RNA Pol II purified from rat liver as described10 is used in this example; however, RNA Pol II purified from other sources, including cultured mammalian cells or yeast can also be used3,4,5,11,12,13.</li>
	</ol>
	</li>
	<li>Complete the elongation complex by addition of biotinylated non-template DNA.
	<ol>
		<li>Add 5 pmol (1 &#181;L) of immobilized non-template DNA oligo to 14 &#181;L of non-template DNA buffer (from Table 4), add to tube&#160;from step 1.3.2, and incubate for 10 min at 37 &#176;C.</li>
		<li>Place the sample in magnetic rack for 2 min. Wash 1x with 30 &#181;L of wash buffer (from Table 7) to remove unincorporated Pol II and oligos.</li>
	</ol>
	</li>
	<li>Generate elongation complexes containing radiolabeled RNA 23mers.
	<ol>
		<li>Perform all further incubations at 30 &#176;C. Add 1 &#181;L of 15 &#181;M ATP and 10 &#181;Ci (1 &#181;L) of [&#945;-32P] UTP (3,000 Ci/mmol) to 23 &#181;L of Pulse buffer and use this to resuspend washed magnetic beads. 
		CAUTION: Radioactive material is hazardous. Make sure to wear appropriate protective equipment and to follow all lab guidelines for safe use and disposal of radioactive material. 
		NOTE: Replace radiolabeled UTP with 15 &#181;M UTP when radiolabeled RNA is not needed, such as for western blot experiments.</li>
		<li>Incubate the reaction for 10 min to allow the synthesis of radiolabeled 23mers.</li>
		<li>Add 1.5 &#181;L of solution containing 100 &#181;M ATP and 100 &#181;M UTP mix to 3.5 &#181;L of chase buffer, add to tube containing preassembled elongation complexes, and incubate for 5 min to chase all nascent transcripts into 23mers.</li>
		<li>Place the sample in magnetic rack for 2 min, remove the supernatant, wash once with 30 &#181;L of wash buffer to remove unincorporated nucleotides, and resuspend in 30 &#181;L of wash buffer.</li>
		<li>Either add 94 &#181;L of stop mix with proteinase K and glycogen (Table 9) to terminate reactions or proceed to section 1.6 to generate elongation complexes with longer transcripts or section 2 to perform capping assays.</li>
	</ol>
	</li>
	<li>(Optional) Walk Pol II to make 23, 25, and 29 nucleotide transcripts
	<ol>
		<li>Follow the procedure described in steps 1.2.1 through 1.5.4&#160;to prepare elongation complexes containing radiolabeled 23mers. Scale up 4-fold to generate 120 &#181;L of washed elongation complexes, which is enough complexes for 4 reactions.</li>
		<li>Label 3 new tubes &#8220;23mer&#8221;, &#8220;25mer&#8221; and &#8220;29mer&#8221;. Transfer 30 &#181;L of washed elongation complexes to &#8220;23mer&#8221; tube and add 94 &#181;L of stop mix with proteinase K and glycogen.</li>
		<li>Place the tube containing the remaining 90 &#181;L of washed elongation complexes in the magnetic rack for 2 min, remove the supernatant, and resuspend beads in 90 &#181;L of BTB supplemented with 1.2 &#181;L each of 1.5 mM ATP and 1.5 mM CTP. Incubate for 10 min at 30 &#176;C.</li>
		<li>After washing once with 90 &#181;L of wash buffer and resuspending in 90 &#956;L of wash buffer as described in step 1.5.4, transfer 30 &#181;L of elongation complexes to &#8220;25mer&#8221; tube and add 94 &#181;L of stop mix with proteinase K and glycogen.</li>
		<li>Place the tube containing the remaining 60 &#181;L of elongation complexes in the magnetic rack for 2 min, remove the supernatant, and resuspend beads in 60 &#181;L of BTB supplemented with 0.8&#160;&#181;L each of 1.5 mM ATP and 1.5 mM CTP.</li>
		<li>Incubate for 10 min at 30 &#176;C, wash once with 60 &#181;L of wash buffer as in section 1.5.5, and resuspend in 60 &#181;L of wash buffer.</li>
		<li>Transfer 30 &#181;L from 2x sample to &#8220;29mer&#8221; tube and either add 94 &#181;L of stop mix with proteinase K and glycogen or proceed to section 2 to perform capping assays.</li>
		<li>Proceed to RNA purification and analysis (Section 3).</li>
	</ol>
	</li>
</ol>
2. Using Artificial Elongation Complexes to Assay Cotranscriptional Capping
<ol>
	<li>Generate washed elongation complexes containing 23mers by scaling up the procedure described in steps 1.2.1 through 1.5.5 13-fold. This is enough for 12 reactions + 1 extra.</li>
	<li>Pol II CTD phosphorylation
	<ol>
		<li>Place the tube containing washed elongation complexes in the magnetic rack for 2 min, remove the supernatant, and resuspend beads in 377&#160;&#181;L of BTB supplemented with 13 &#181;L of 1.5 mM ATP.</li>
		<li>Prepare two new tubes, labeled +H and -H, respectively. To the tube labeled +H add 3 &#181;L of ~300 ng/&#181;L TFIIH, and to the tube labeled -H add 9 &#181;L of 20&#8201;mM HEPES-NaOH pH 7.9, 20% glycerol, 100&#8201;mM KCl, 1&#8201;mM EDTA, 0.5&#8201;mg/mL bovine serum albumin. 
		NOTE: We typically use TFIIH purified from rat liver, but we have obtained similar results using ~0.6 &#181;g/reaction of commercially-available recombinant Cdk7/Cyclin H/MAT1 (CAK, which is the kinase module from TFIIH). See Table of Materials for information.</li>
		<li>Add 87&#160;&#181;L of the washed elongation complexes from step 2.2.1 to the tube labeled +H and 270 &#181;L of washed elongation complexes to the tube labeled -H. Incubate 10 min at 30 &#176;C.</li>
		<li>Place sample in magnetic rack for 2 min. To remove excess nucleotides and unbound protein, wash elongation complexes in +H and -H reactions with 90 &#181;L or 270 &#181;L of wash buffer, respectively.</li>
	</ol>
	</li>
	<li>RNA capping
	<ol>
		<li>Resuspend elongation complexes in the tube labeled +H in 87 &#181;L of capping mix (BTB supplemented with 50 &#181;M GTP (Table 10). Resuspend elongation complexes in the tube labeled -H in 261&#160;&#181;L of capping mix.</li>
		<li>Prepare 4 new tubes labeled 5 ng CE+H, 5 ng CE, 15 ng CE, 45 ng CE. Dilute capping enzyme (CE) into 20&#8201;mM HEPES-NaOH pH 7.9, 20% glycerol, 100&#8201;mM KCl, 1&#8201;mM EDTA, 0.5&#8201;mg/mL bovine serum albumin to prepare solutions containing 5 ng CE/&#181;L, 15 ng CE/&#181;L, or 45 ng CE/&#181;L and dispense 3 &#181;L of the appropriate solution into each of the 4 tubes.</li>
		<li>Prepare 12 new tubes with 94 &#181;L of stop buffer (2.1.1) added to each.</li>
		<li>Start each capping reaction by adding 87 &#181;L of elongation complex treated with TFIIH or not to labeled tubes containing capping enzyme. Incubate at 30 &#176;C in a heat block.</li>
		<li>After 1, 2, or 4 min, stop reactions by transferring 30 &#181;L of each reaction mixture to tubes containing 94 &#181;L of stop buffer. Purify and analyze reaction products as described in section 3.</li>
	</ol>
	</li>
</ol>
3. RNA Purification and Analysis
<ol>
	<li>Purify RNA
	<ol>
		<li>Incubate reaction products in stop buffer for 20 min at room temperature. 
		NOTE: Pausing point. Samples in this buffer has been maintained for up to 4 h without loss or degradation of RNA.</li>
		<li>Extract once with 124 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1). 
		CAUTION: Phenol is extremely toxic and is rapidly absorbed by the skin. Wear appropriate protective equipment. 
		NOTE: To maximize recovery of RNA during extraction, use commercially available tubes containing a high-density gel, which forms a stable barrier between aqueous and organic phases. See Discussion and Table of Materials.</li>
		<li>Transfer the aqueous phase (top layer) to new tubes containing 12.4 &#181;L of 3 M sodium acetate pH 5.2.</li>
	</ol>
	</li>
	<li>Ethanol precipitation
	<ol>
		<li>Add 350 &#181;L of 100% ethanol and mix thoroughly by inversion.</li>
		<li>Incubate for at least 10 min on dry ice. 
		NOTE: Pausing point. For convenience, one can stop here and complete the procedure on the next day; however, it is possible to proceed directly to step 3.3 and run the gel on the same day. RNA can be kept at -80 &#176;C for a couple of days before further processing, but do not leave for longer times.</li>
	</ol>
	</li>
	<li>Preparing RNA samples for gel electrophoresis
	<ol>
		<li>Allow samples to thaw, mix by inverting 2-3 times, and centrifuge for 15 min at 21,000 x g, 4 &#176;C, in a tabletop centrifuge. Meanwhile, set up denaturing gel mix (section 3.4.1).</li>
		<li>Carefully remove ethanol from samples without disturbing pellet.</li>
		<li>Add 500 &#181;L of 70% ethanol, invert tube a couple of times to wash pellet, and then centrifuge again at 21,000 x g for 5 min at either room temperature or 4 &#176;C.</li>
		<li>Remove as much ethanol as possible and do a quick spin (5-10 s) of the tubes in a table top centrifuge. Then, with a gel-loading tip, remove the last remaining volume of ethanol.</li>
		<li>Air-dry pellet for 3 min at room temperature.</li>
		<li>Dissolve RNA by adding 4 &#181;L of H2O to the top of each pellet. Incubate 5 min at room temperature.</li>
		<li>Add 4 &#181;L of 2x RNA dye (See Table of Materials) to the mix, and then vortex each tube for a couple of seconds to fully resuspend RNA. 
		NOTE: RNA dye that contains formamide instead of urea is recommended, since the latter precipitates at low temperatures.</li>
		<li>Quick-spin the tubes, and then incubate them at 70 &#176;C for 10 min in a heat block.</li>
		<li>Store tubes on dry ice until ready to load on gel. 
		NOTE: Keeping RNA on dry ice allows the flexibility to work on other experiments while the gel is polymerizing. Samples do not need to be reheated after thawing. However, RNA can be directly loaded after heating if the gel is ready to run.</li>
	</ol>
	</li>
	<li>Preparing Urea-PAGE gel
	<ol>
		<li>For 40 mL gel mix, combine in a 50 mL conical tube: 16.7 g of urea, 15 mL of 40% bis:acrylamide solution (19:1 ratio), 4 mL of 10x TBE (1 M Tris-HCl, 1 M sodium borate, 20 mM EDTA), and 8 mL of H2O. This volume is sufficient for one standard size gel (18 cm height x 16 cm width) with 1.0 mm spacers. 
		CAUTION: Acrylamide is extremely toxic. While there is no inhalation risk when it is in solution, always wear lab coat, gloves, and safety glasses while handling. 
		NOTE: This mixture is for casting a denaturing 15% polyacrylamide gel, which readily resolves RNA between 15-50 nt. Change the concentration of bis/acrylamide as needed to resolve different RNA transcripts of different lengths.</li>
		<li>Place closed tube containing gel mix on a nutator until urea is fully dissolved.</li>
		<li>Set up gel-casting station with glass plates, 1 mm spacers and a 15-well comb.</li>
		<li>Working rapidly, add 40 &#181;L of TEMED and 400 &#181;L of 10% ammonium persulfate solution to gel solution and mix well. Using a 25 mL pipet, pour gel solution between plates, insert comb, and allow the gel to polymerize for at least 2 h. 
		NOTE: If the gel is poured more than 2 h before use, once it has polymerized cover it with moist paper towel (leaving the comb in place) and wrap with plastic wrap to prevent it from drying out.</li>
	</ol>
	</li>
	<li>Denaturing gel RNA electrophoresis
	<ol>
		<li>After polymerization, transfer gel to running tank and pre-run it at 20 mA in 1x TBE for 15-30 min.</li>
		<li>Meanwhile, thaw samples (if frozen), vortex, and spin them for 4 min at 2,000 x g, 4 &#176;C.</li>
		<li>When ready, turn off the power supply, and carefully rinse each well with 1x TBE using a syringe.</li>
		<li>Use gel-loading tips to load samples onto the gel.</li>
		<li>Run gel at a constant 20-30 mA for about 2 h or until lower dye (xylene cyanol FF) reaches the bottom of the gel.</li>
	</ol>
	</li>
	<li>Remove the gel, place it on a piece of absorbent paper, and wrap with plastic wrap.</li>
	<li>Expose the radiolabeled gel to a phosphorscreen.</li>
	<li>Scan phosphorscreen using a phosphorimager and analyze image.</li>
</ol>

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The authors have nothing to disclose.

Studies that seek to dissect events coupled to the Pol II elongation complex such as RNA processing and regulation of the transcript elongation itself can be greatly facilitated by use of a highly purified enzyme system. Setting up such enzyme systems can be challenging. Promoter-dependent transcription by Pol II requires at least five general transcription factors. Preparing and stockpiling these factors can take months; hence, the rate-limiting step in this process is often simply preparing the cadre of transcription f...

Eukaryotic messenger RNA (mRNA) synthesis is an elaborate biochemical process that involves synthesis of an unprocessed precursor RNA by RNA polymerase II and processing of the precursor RNA to yield the mature mRNA. The RNA processing steps of capping, splicing, and polyadenylation are carried out largely co-transcriptionally. The Pol II elongation complex serves as a scaffold that recruits and orchestrates the activities of many of the RNA processing enzymes. Consequently, our ultimate understanding of how mature eukaryotic mRNAs are generated will rely heavily on the development of experimental systems to allow dissection of the biochemical mechanisms underlying recruitment to the elongation complex and regulation of enzymes responsible for co-transcriptional capping, splicing, and polyadenylation.
Not surprisingly, development of such experimental systems has been difficult. A major impediment has been the remarkable complexity of Pol II transcription itself where simply reconstituting basal transcription by Pol II in vitro requires a minimum set of five general transcription initiation factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH1. Moreover, reconstituting any sort of regulated Pol II transcription in vitro requires an even larger set of transcription factors and coregulators. Thus, a major goal has been to develop simpler experimental systems allowing reconstitution of active Pol II elongation complexes suitable for investigations of the functional coupling of Pol II transcription and RNA processing.
One such simpler method for reconstituting active Pol II elongation complexes has proven useful for structural and biochemical studies of elongating Pol II and, more recently, for investigating co-transcriptional RNA processing2,3,4,5. In this article, we show how Pol II elongation complexes prepared from purified Pol II and synthetic transcription bubbles can be used effectively to investigate the mechanisms underlying co-transcriptional capping of nascent Pol II transcripts.
Capping refers to the covalent addition of a 5&#8217;-guanosine &#8220;cap&#8221; to the 5&#8217;-triphosphate end of nascent Pol II transcripts. The cap is important for subsequent steps of mRNA maturation, transport, translation, and other processes6,7. The cap is added co-transcriptionally to Pol II transcripts by an enzyme referred to as capping enzyme. In mammalian cells, active sites responsible for the RNA 5&#8217;-triphosphatase and guanylyl transferase activities of the capping enzyme are contained within a single polypeptide8. The capping enzyme is recruited to the Pol II elongation complex through interactions with yet to be defined surfaces on the Pol II body and the Rpb1 carboxy-terminal domain (CTD) phosphorylated on Ser5 of its heptapeptide repeats5. In the elongation complex, the capping enzyme catalyzes addition of a 5&#8217;-guanosine cap once the nascent transcript reaches a length of at least 18 nucleotides and has emerged from the polymerase RNA exit channel. In the first step of the capping reaction, the triphosphatase hydrolyzes the RNA 5&#8217;-triphosphate to yield a 5&#8217;-diphosphate. In the second step, GTP is hydrolyzed to GMP by the guanylyl transferase, forming a GMP-capping enzyme intermediate. Finally, the guanylyl transferase transfers GMP to the 5&#8217;-diphosphate end of the nascent transcript to produce the cap.
A remarkable feature of the capping reaction is that co-transcriptional capping (i.e., capping of transcripts associated with functional Pol II elongation complexes) is much more efficient than capping of free RNA5,9. Thus, a major question in the field has been how this dramatic activation of capping is achieved via interactions of the capping enzyme with the Pol II elongation complex. In this protocol we describe the assembly of active RNA polymerase II elongation complexes using only purified RNA polymerase II and artificial transcription bubbles. These methods allow creation of RNA polymerase II elongation complexes with transcripts of defined length and sequence. In a recent study, we used these defined RNA polymerase II elongation complexes as a model for investigating aspects of the mechanisms of RNA capping5. In particular, we showed that (i) capping of RNA associated with these elongation complexes was more than 100-fold more efficient than capping of free RNA and (ii) was stimulated by TFIIH-dependent phosphorylation of the Pol II CTD. The approach described here could in principle be adapted to generate substrates for studying other co-transcriptional RNA processing reactions linked to the Pol II elongation complex.
In Section 1 of this protocol, artificial elongation complexes are created by annealing a synthetic template strand DNA oligonucleotide to an RNA oligonucleotide that is complementary at its 3&#8217;-end to approximately 9 nucleotides of the template strand DNA. Pol II is then loaded onto the DNA:RNA duplex. The elongation complex is then completed by addition of a partially complementary, non-template strand DNA oligonucleotide that is labeled with biotin at its 3&#8217;-end (Figure 1 and Figure 2A). The RNA oligonucleotide is extended by Pol II in these elongation complexes to make radiolabeled transcripts of defined length and sequence upon addition of appropriate combinations of radiolabeled nucleotides. In addition, using a combination of washes to remove unincorporated nucleotides and further addition of different combinations of nucleotides, one can &#8220;walk&#8221; Pol II to different positions along the DNA template and synthesize RNA of defined lengths. RNA is then purified and subjected to electrophoresis in denaturing urea-PAGE gels. In Section 2 of the protocol, artificial elongation complexes are used to analyze co-transcriptional RNA capping. The example presented measures the effect of TFIIH-dependent phosphorylation of the Pol II CTD on co-transcriptional RNA capping. In this experiment, we measure the extent of co-transcriptional capping as a function of capping enzyme concentration (5, 15 and 45 ng per reaction) and time (1, 2 and 4 min).

<table>
	<tbody>
		<tr>
			<td>[&#945;-32P] UTP (3000 Ci/mmol), 1 mCi</td>
			<td>Perkin Elmer</td>
			<td>NEG007H001MC</td>
			<td>For radiolabeling RNA</td>
		</tr>
		<tr>
			<td>2x RNA loading dye</td>
			<td>New England Biolabs</td>
			<td>B0363S</td>
			<td>Highly recommended. For preparing RNA during gel loading</td>
		</tr>
		<tr>
			<td>40% Bis:Acrylamide solution</td>
			<td>Biorad</td>
			<td>1610144</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (20 mg/ml)</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk7/Cyclin H/MAT1 (CAK complex) Protein, active, 10 &#181;g</td>
			<td>Millipore Sigma</td>
			<td>14-476</td>
			<td>Used to phosphorylate Pol II CTD</td>
		</tr>
		<tr>
			<td>DNA oligonucleotides</td>
			<td>IDT</td>
			<td></td>
			<td>See Table 8 for purity specifications</td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1</td>
			<td>Life Technologies Invitrogen</td>
			<td>65001</td>
			<td>We have also used Dynabeads M-280 streptavidin without problem but prefer MyOne Streptavidin beads because they sediment more slowly</td>
		</tr>
		<tr>
			<td>GlycoBlue Coprecipitant (15&#8201;mg/ml)</td>
			<td>Life Technologies Invitrogen</td>
			<td>AM9516</td>
			<td>Highly recommended. Dyes nucleic-acid pellet blue making reactions much easier to handle</td>
		</tr>
		<tr>
			<td>Hoefer SE600 standard dual cooled vertical electrophoresis system with 1 mm spacers and 15 well comb</td>
			<td>Hoefer</td>
			<td>SE600-15-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>MaXtract high density tubes (1.5 ml)</td>
			<td>Qiagen</td>
			<td>129046</td>
			<td>Highly recommended. Contains a high density gel that forms a stable barrier between aqueous and organic phases; improves RNA yields during extractions</td>
		</tr>
		<tr>
			<td>Proteinase K solution (20&#8201;mg/ml)</td>
			<td>Life Technologies Invitrogen</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA oligonucleotides</td>
			<td>Trilink</td>
			<td></td>
			<td>See Table 8 for more details</td>
		</tr>
		<tr>
			<td>Yeast Inorganic Pyrophosphatase (100 units/ml)</td>
			<td>New England Biolabs</td>
			<td>NEBM2403S</td>
			<td>Required only during capping reactions</td>
		</tr>
	</tbody>
</table>

artificial rna polymerase ii elongation complexes for dissecting co-transcriptional rna processing events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Figures 2 and 3 show representative result reactions used to generate artificial elongation complexes containing transcripts of different lengths by extending or Pol II from different sources. Figure 4 depicts how these elongation complexes can be used to assay co-transcriptional CTD phosphorylation-dependent RNA capping.
Figure 2A is a...

Watch this Scientific Journal Video about Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events at JoVE.com

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

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9.6K Views.  Stowers Institute for Medical Research. Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Video: Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...