Name: artificial rna polymerase ii elongation complexes for dissecting co-transcriptional rna processing events
Uploaded: 2019-05-13T14:00:00.000+00:00
Duration: 10 min 59 s
Description: Watch this Scientific Journal Video about Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events at JoVE.com

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>[&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P] 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 &amp;micro;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&amp;thinsp;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&amp;thinsp;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...

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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

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Biochemistry

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) following a head-on collision is unknown. Here, we find that the E. coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also find that the transcription-repair coupling factor, Mfd, promotes direct restart of the fork following the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a novel role for the transcription-coupled repair pathway in promoting replication past a RNAP block.

The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

Biology

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

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

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

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Bioengineering

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Eukaryotic RNA Polymerases

RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the TATA-binding protein is common to all. These proteins remain attached to the RNAP to guide the direction of RNA synthesis on the template DNA strand. Once RNA strand elongation is complete, the RNAP and associated proteins need to disassemble and release the mRNA transcript.
Unlike the termination signals encoded by bacterial genes, the protein-encoding genes transcribed by RNA Polymerase II lack specific sequences that direct the enzyme to terminate at precise locations. The most common termination pathway, known as the Poly(A) dependent termination, combines polyadenylation of the mRNA transcript with RNAP termination. Here, while the RNA Polymerase II continues to transcribe RNA, sometimes up to thousands of basepairs past the end of the gene sequence, the transcript is cleaved at an internal site. Thus the upstream part of the transcript is released and a polyadenine tail can be added to the 3&#39; end of the cleaved transcript. The downstream cleavage product is digested by a 5&#39;-exonuclease while it is still being transcribed by the RNA Polymerase II. When the 5&#39;-exonuclease digests all of the remainder transcripts, it helps the RNAP to dissociate from its DNA template strand, thus completing the transcription.

The Three Eukaryotic RNA Polymerases

Polimerazy eukariotycznego RNA

RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional ...

KEY TERMS AND CONCEPTS

RNA Polymerase II Accessory Proteins

Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in prokaryotes, the catabolite activator protein or CAP directly interacts with the C-terminal domain of the alpha subunit of RNA polymerase to regulate gene expression. Strong evidence for direct interaction is the loss of function mutations in the activation domains of proteins that lead to suppression of transcriptional activity.
However, in some eukaryotic genes, regulation can happen via distal activation. Hence, the regulating elements may not lie in close proximity to the promoter or may not interact directly with the transcriptional machinery. Such interactions can be detected by (a) observing the rate of transcription in the presence or absence of the regulatory protein (b) mutations in the binding site of the regulatory protein that can disrupt gene expression (c)&#160; measuring the binding affinity between the regulatory protein and the promoter.
In addition, the transcription machinery also needs nucleosome remodelers to access the DNA within the chromatin. Hence, these nucleosome remodelers are also involved in regulating gene expression.

Białka pomocnicze polimerazy II RNA

Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, ...

Transcription Elongation Factors

Transcription elongation is a dynamic process that alters depending upon the sequence heterogeneity of the DNA being transcribed. Hence, it is not surprising that the elongation complex&#39;s composition also varies along the way while transcribing a gene.
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a protein. However, in eukaryotes, the transcription is coupled with mRNA processing. Hence, pausing of RNA polymerase around exon-intron junctions is necessary for increasing the efficiency of mRNA splicing.
These halts in RNA Polymerase activity may be reversible or irreversible. In case of a reversible pause, proteins such as TFIIF, elongins, ELL, ensure that the RNA Polymerase resumes elongation after a brief pause. However, if the halt in RNA Polymerase activity is irreversible, it becomes a transcriptional arrest. If transcription is arrested, then the enzyme cannot resume elongation on its own. In such a situation, elongation factors such as TFIIS and pTEFb enable RNA Polymerase II to read through the DNA template at transcriptional arrest sites.
In addition, ATP-dependent chromatin remodeling factors and histone chaperones are also involved in the regulation of transcription elongation. Together they can alter the positions of nucleosomes along the DNA, making it accessible or inaccessible to the transcription machinery.
Hence, RNA polymerase needs the help of several factors to cruise through chromatin and specific sequences that interfere with transcription.

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

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Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

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

High-throughput Purification of Affinity-tagged Recombinant Proteins

Chemical Triphosphorylation of Oligonucleotides

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Polimerazy eukariotycznego RNA

Białka pomocnicze polimerazy II RNA

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