Name: an assay for quantifying protein-rna binding in bacteria
Uploaded: 2019-06-12T14:00:00
Description: Watch this Scientific Journal Video about An Assay for Quantifying Protein-RNA Binding in Bacteria at JoVE.com

This project received funding from the I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation (Grant No. 152/11), Marie Curie Reintegration Grant No. PCIG11-GA- 2012-321675, and from the European Union&#39;s Horizon 2020 Research and Innovation Program under grant agreement no. 664918 - MRG-Grammar.

1. System Preparation
<ol>
	<li>Design of binding-site plasmids 
	<ol>
		<li>Design the binding site cassette as depicted in Figure 1. Each minigene contains the following parts (5&#39; to 3&#39;): Eagl restriction site, &#8764;40 bases of the 5&#39; end of the kanamycin (Kan) resistance gene, pLac-Ara promoter, ribosome binding site (RBS), AUG of the mCherry gene, a spacer (&#948;), an RBP binding site, 80 bases of the 5&#39; end of the mCherry gene, and an ApaLI restrict...

<ol>
	<li>Cerretti, D. P., Mattheakis, L. C., Kearney, K. R., Vu, L., Nomura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+regulation+of+the+spc+operon+in+Escherichia+coli.+Identification+and+structural+analysis+of+the+target+site+for+S8+repressor+protein.">Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein.</a> Journal of Molecular Biology. 204 (2), 309-329 (1988).</li><li>Babitzke, P., Baker, C. S., Romeo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+translation+initiation+by+RNA+binding+proteins.">Regulation of translation initiation by RNA binding proteins.</a> Annual Review of Microbiology. 63, 27-44 (2009).</li><li>Van Assche, E., Van Puyvelde, S., Vanderleyden, J., Steenackers, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+involved+in+post-transcriptional+regulation+in+bacteria.">RNA-binding proteins involved in post-transcriptional regulation in bacteria.</a> Frontiers in Microbiology. 6, 141 (2015).</li><li>Chappell, J., Watters, K. E., Takahashi, M. K., Lucks, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+renaissance+in+RNA+synthetic+biology:+new+mechanisms,+applications+and+tools+for+the+future.">A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future.</a> Current Opinion in Chemical Biology. 28, 47-56 (2015).</li><li>Wagner, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule-based+regulation+of+RNA-delivered+circuits+in+mammalian+cells.">Small-molecule-based regulation of RNA-delivered circuits in mammalian cells.</a> Nature Chemical Biology. 14 (11), 1043 (2018).</li><li>Bendak, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+assessing+the+RNA-binding+potential+of+a+protein.">A rapid method for assessing the RNA-binding potential of a protein.</a> Nucleic Acids Research. 40 (14), e105 (2012).</li><li>Strein, C., Alleaume, A. -. M., Rothbauer, U., Hentze, M. W., Castello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+assay+for+RNA-binding+proteins+in+living+cells.">A versatile assay for RNA-binding proteins in living cells.</a> RNA. 20 (5), 721-731 (2014).</li><li>Ule, J., Jensen, K. B., Ruggiu, M., Mele, A., Ule, A., Darnell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP+identifies+Nova-regulated+RNA+networks+in+the+brain.">CLIP identifies Nova-regulated RNA networks in the brain.</a> Science. 302 (5648), 1212-1215 (2003).</li><li>Lee, F. C. Y., Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+CLIP+Technologies+for+Studies+of+Protein-RNA+Interactions.">Advances in CLIP Technologies for Studies of Protein-RNA Interactions.</a> Molecular Cell. 69 (3), 354-369 (2018).</li><li>Katz, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+Vivo+Binding+Assay+for+RNA-Binding+Proteins+Based+on+Repression+of+a+Reporter+Gene.">An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene.</a> ACS Synthetic Biology. 7 (12), 2765-2774 (2018).</li><li>Watters, K. E., Yu, A. M., Strobel, E. J., Settle, A. H., Lucks, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+RNA+structures+in+vitro+and+in+vivo+with+selective+2&#8217;-hydroxyl+acylation+analyzed+by+primer+extension+sequencing+(SHAPE-Seq).">Characterizing RNA structures in vitro and in vivo with selective 2&#8217;-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).</a> Methods. 103, 34-48 (2016).</li><li>Saito, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+translational+regulation+by+an+L7Ae-kink-turn+RNP+switch.">Synthetic translational regulation by an L7Ae-kink-turn RNP switch.</a> Nature Chemical Biology. 6 (1), 71-78 (2010).</li><li>Gott, J. M., Wilhelm, L. J., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+properties+of+the+coat+protein+from+bacteriophage+GA.">RNA binding properties of the coat protein from bacteriophage GA.</a> Nucleic Acids Research. 19 (23), 6499-6503 (1991).</li><li>Peabody, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+binding+site+of+bacteriophage+MS2+coat+protein.">The RNA binding site of bacteriophage MS2 coat protein.</a> The EMBO Journal. 12 (2), 595-600 (1993).</li><li>Lim, F., Peabody, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+recognition+site+of+PP7+coat+protein.">RNA recognition site of PP7 coat protein.</a> Nucleic Acids Research. 30 (19), 4138-4144 (2002).</li><li>Lim, F., Spingola, M., Peabody, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA-binding+Site+of+Bacteriophage+Q&#946;+Coat+Protein.">The RNA-binding Site of Bacteriophage Q&#946; Coat Protein.</a> Journal of Biological Chemistry. 271 (50), 31839-31845 (1996).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>. Optimizing Restriction Endonuclease Reactions Available from: <a target="_blank" href="https://international.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions">https://international.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions</a> (2018)</li><li>. Wizard&#174; SV Gel and PCR Clean-Up System Protocol Available from: <a target="_blank" href="https://worldwide.promega.com/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/">https://worldwide.promega.com/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/</a> (2018)</li><li>. Ligation Protocol with T4 DNA Ligase (M0202) Available from: <a target="_blank" href="https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202">https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202</a> (2018)</li><li>. Routine Cloning Using Top10 Competent Cells - US Available from: <a target="_blank" href="https://www.thermofisher.com/us/en/home/references/protocols/cloning/competent-cells-protocol/routine-cloning-using-top10-competent-cells.html">https://www.thermofisher.com/us/en/home/references/protocols/cloning/competent-cells-protocol/routine-cloning-using-top10-competent-cells.html</a> (2018)</li><li>. NucleoSpin Plasmid - plasmid Miniprep kit Available from: <a target="_blank" href="https://www.mn-net.com/ProductsBioanalysis/DNAandRNApurification/PlasmidDNApurificationeasyfastreliable/NucleoSpinPlasmidplasmidMiniprepkit/tabid/1379/language/en-US/Default.aspx">https://www.mn-net.com/ProductsBioanalysis/DNAandRNApurification/PlasmidDNApurificationeasyfastreliable/NucleoSpinPlasmidplasmidMiniprepkit/tabid/1379/language/en-US/Default.aspx</a> (2018)</li><li>Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., Roe, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+in+single-stranded+bacteriophage+as+an+aid+to+rapid+DNA+sequencing.">Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing.</a> Journal of Molecular Biology. 143 (2), 161-178 (1980).</li><li>. Protocol - How to Create a Bacterial Glycerol Stock Available from: <a target="_blank" href="https://www.addgene.org/protocols/create-glycerol-stock/">https://www.addgene.org/protocols/create-glycerol-stock/</a> (2018)</li><li>. Making your own chemically competent cells Available from: <a target="_blank" href="https://international.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells">https://international.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells</a> (2018)</li><li>. Luria-Bertani (LB) Medium Preparation &#183; Benchling Available from: <a target="_blank" href="https://benchling.com/protocols/gdD7XI0J/luria-bertani-lb-medium-preparation">https://benchling.com/protocols/gdD7XI0J/luria-bertani-lb-medium-preparation</a> (2018)</li><li>Delebecque, C. J., Silver, P. A., Lindner, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+and+using+RNA+scaffolds+to+assemble+proteins+in+vivo.">Designing and using RNA scaffolds to assemble proteins in vivo.</a> Nature Protocols. 7 (10), 1797-1807 (2012).</li><li>Hocine, S., Raymond, P., Zenklusen, D., Chao, J. A., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+gene+expression+using+two-color+RNA+labeling+in+live+yeast.">Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.</a> Nature Methods. 10 (2), 119-121 (2013).</li><li>Espah Borujeni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+quantification+of+translation+inhibition+by+mRNA+structures+that+overlap+with+the+ribosomal+footprint+in+N-terminal+coding+sequences.">Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences.</a> Nucleic Acids Research. 45 (9), 5437-5448 (2017).</li><li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome-wide+profiling+of+RNA+secondary+structure+reveals+novel+regulatory+features.">In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.</a> Nature. 505, (2013).</li><li>Rouskin, S., Zubradt, M., Washietl, S., Kellis, M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+probing+of+RNA+structure+reveals+active+unfolding+of+mRNA+structures+in+vivo.">Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo.</a> Nature. 505 (7485), 701-705 (2014).</li><li>Lucks, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+RNA+structure+characterization+with+selective+2&#8217;-hydroxyl+acylation+analyzed+by+primer+extension+sequencing+(SHAPE-Seq).">Multiplexed RNA structure characterization with selective 2&#8217;-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), 11063-11068 (2011).</li><li>Spitale, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+imprints+in+vivo+decode+RNA+regulatory+mechanisms.">Structural imprints in vivo decode RNA regulatory mechanisms.</a> Nature. 519 (7544), 486 (2015).</li><li>Watters, K. E., Abbott, T. R., Lucks, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+characterization+of+cellular+RNA+structure+and+function+with+in-cell+SHAPE-Seq.">Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.</a> Nucleic Acids Research. 44 (2), e12 (2016).</li><li>Flynn, R. 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The method described in this article facilitates quantitative in vivo measurement of RBP-RNA binding affinity in E. coli cells. The protocol is relatively easy and can be conducted without the use of sophisticated machinery, and data analysis is straightforward. Moreover, the results are produced immediately, without the relatively long wait-time associated with next generation sequencing (NGS) results.
One limitation to this method is that it works only in bacterial cells. However, a...

RNA binding protein (RBP)-based post-transcriptional regulation, specifically characterization of the interaction between RBPs and RNA, has been studied extensively in recent decades. There are multiple examples of translational down-regulation in bacteria originating from RBPs inhibiting, or directly competing with, ribosome binding1,2,3. In the field of synthetic biology, RBP-RNA interactions are emerging as a significant tool for the design of transcription-based genetic circuits4,5. Therefore, there is an increase in demand for characterization of such RBP-RNA interactions in a cellular context.
The most common methods for studying protein-RNA interactions are the electrophoretic mobility shift assay (EMSA)6, which is limited to in vitro settings, and various pull-down assays7, including the CLIP method8,9. While such methods enable the discovery of de novo RNA binding sites, they suffer from drawbacks such as labor-intensive protocols and expensive deep sequencing reactions and may require a specific antibody for RBP pull-down. Due to the susceptible nature of RNA to its environment, many factors can affect RBP-RNA interactions, emphasizing the importance of interrogating RBP-RNA binding in the cellular context. For example, we and others have demonstrated significant differences between RNA structures in vivo and in vitro10,11.
Based on the approach of a previous study12, we recently demonstrated10 that when placing pre-designed binding sites for the capsid RBPs from the bacteriophages GA13, MS214, PP715, and Q&#946;16 in the translation initiation region of a reporter mRNA, reporter expression is strongly repressed. We present a relatively simple and quantitative method, based on this repression phenomenon, to measure the affinity between RBPs and their corresponding RNA binding sites in vivo.

<table>
	<tbody>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>SIGMA</td>
			<td>A9518</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>ALFA AESAR</td>
			<td>33337</td>
			<td></td>
		</tr>
		<tr>
			<td>48 plates</td>
			<td>Axygen</td>
			<td>P-5ML-48-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>8- lane plates</td>
			<td>Axygen</td>
			<td>RESMW8I</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Axygen</td>
			<td>P-DW-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates for plate reader</td>
			<td>Perkin Elmer</td>
			<td>6005029</td>
			<td></td>
		</tr>
		<tr>
			<td>ApaLI</td>
			<td>NEB</td>
			<td>R0507</td>
			<td></td>
		</tr>
		<tr>
			<td>Binding site sequences</td>
			<td>Gen9 Inc. and Twist Bioscience</td>
			<td></td>
			<td>see Table 1</td>
		</tr>
		<tr>
			<td>E. coli TOP10 cells</td>
			<td>Invitrogen</td>
			<td>C404006</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagl-HF</td>
			<td>NEB</td>
			<td>R3505</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>BIO LAB</td>
			<td>071205</td>
			<td></td>
		</tr>
		<tr>
			<td>incubator</td>
			<td>TECAN</td>
			<td>liconic incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin solfate</td>
			<td>SIGMA</td>
			<td>K4000</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI- HF</td>
			<td>NEB</td>
			<td>R0142</td>
			<td></td>
		</tr>
		<tr>
			<td>ligase</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid-handling robotic system</td>
			<td>TECAN</td>
			<td>EVO 100, MCA 96-channel</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab analysis software</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>multi- pipette 8 lanes</td>
			<td>Axygen</td>
			<td>BR703710</td>
			<td></td>
		</tr>
		<tr>
			<td>N-butanoyl-L-homoserine lactone (C4-HSL)</td>
			<td>cayman</td>
			<td>K40982552 019</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Biological Industries</td>
			<td>020235A</td>
			<td></td>
		</tr>
		<tr>
			<td>platereader</td>
			<td>TECAN</td>
			<td>Infinite F200 PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 HotStart Polymerase</td>
			<td>NEB</td>
			<td>M0493</td>
			<td></td>
		</tr>
		<tr>
			<td>RBP seqeunces</td>
			<td>Addgene</td>
			<td>27121 &#38; 40650</td>
			<td>see Table 2</td>
		</tr>
		<tr>
			<td>SODIUM CHLORIDE (NaCL)</td>
			<td>BIO LAB</td>
			<td>190305</td>
			<td></td>
		</tr>
		<tr>
			<td>SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The presented method utilizes the competition between an RBP and the ribosome for binding to the mRNA molecule (Figure 1). This competition is reflected by decreasing mCherry levels as a function of increased production of RBP-mCerulean, due to increasing concentrations of inducer. In the case of increasing mCerulean fluorescence, with no significant changes in mCherry, a lack of RBP binding is deduced. Representative results for both a positive and a negativ...

Watch this Scientific Journal Video about An Assay for Quantifying Protein-RNA Binding in Bacteria at JoVE.com

An Assay for Quantifying Protein-RNA Binding in Bacteria (Video) | JoVE

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.

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 ...

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Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes (Video) | JoVE

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Quantifying Abdominal Pigmentation in Drosophila melanogaster (Video) | JoVE

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

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

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells (Video) | JoVE

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

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation (Video) | JoVE

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria (Video) | JoVE

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells (Video) | JoVE

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...