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

该项目得到了规划和预算委员会和以色列科学基金会(第152/11号赠款)的I-CORE方案的资助,玛丽·居里重返社会赠款号为第152/11号赠款。PCIG11-GA- 2012-321675,以及欧盟地平线2020年研究与创新计划,根据授权协议664918 - MRG-Grammar。

1. 系统准备
<ol><li>结合位点质粒的设计
	<ol>
<li>如图1所示,设计装订位盒。每个微基因包含以下部分(5'至3'):Eagl限制位点,[40基端的kanamycin(Kan)抗性基因,pLac-Ara启动子,核糖体结合位点(RBS),MCherry基因的AUG,一个间隔(+),一个RBP结合位点,5'端的80个碱基mCherry基因和ApaLI限制位。 注:为了提高测定的成功率,为每个装订位点设计三个结合位盒,垫块至少由一个、...

<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. 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=Transcriptome-wide+interrogation+of+RNA+secondary+structure+in+living+cells+with+icSHAPE.">Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE.</a> Nature Protocols. 11 (2), 273-290 (2016).</li><li>Bernardi, A., Spahr, P. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+Sequence+at+the+Binding+Site+for+Coat+Protein+on+RNA+of+Bacteriophage+R17.">Nucleotide Sequence at the Binding Site for Coat Protein on RNA of Bacteriophage R17.</a> Proceedings of the National Academy of Sciences of the United States of America. 69 (10), 3033-3037 (1972).</li></ol>

本文描述的方法有助于对大肠杆菌细胞中的RBP-RNA结合亲和力进行定量的体内测量。该协议相对简单,无需使用精密机械即可进行,数据分析也非常简单。此外,结果会立即产生,没有与下一代测序 (NGS) 结果相关的相对长的等待时间。
这种方法的一个限制是,它只在细菌细胞中工作。然而,先前的研究12已经证明在哺乳动物细胞中使用类似的方法对L7AE RBP具有?...

近几十年来,基于RNA结合蛋白(RBP)的转录后调节,特别是RPS和RNA之间相互作用的表征,得到了广泛的研究。在来自限制性商业惯例的细菌中,有许多转化降调节的例子,它们抑制或直接与核糖体结合1、2、3竞争。在合成生物学领域,RBP-RNA相互作用正在成为设计基于转录基因电路4,5的重要工具。因此,在细胞环境中对这种RBP-RNA相互作用的表征需求增加。
研究蛋白质-RNA相互作用的最常见方法是电泳移动移位测定(EMSA)6,仅限于体外设置,以及各种下拉测定7,包括CLIP方法8,9.虽然这些方法能够发现脱新RNA结合位点,但它们存在诸如劳动密集型协议和昂贵的深度测序反应等缺点,并且可能需要一种特定的抗体来进行RBP下拉。由于RNA对其环境的易感性,许多因素会影响RBP-RNA相互作用,强调在细胞环境中对RBP-RNA结合进行探究的重要性。例如,我们和其他人已经证明体内RNA结构与体外RNA结构有显著差异10,11。
根据先前研究12的方法,我们最近演示了10,当为来自噬菌体GA 13、MS2 14、PP715和Q+16的细胞内大黄蜂RBPs放置预先设计的结合位点时,翻译启动区一位记者mRNA,记者的表情被强烈压制。基于这种抑制现象,我们提出了一种相对简单和定量的方法,以测量RBPs与其在体内的相应RNA结合位点之间的亲和力。

<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

在蛋白质转化的起始步骤中,核糖体与mRNA的起始区域结合。通过将RNA结合蛋白(RBP)与mRNA的起始区域结合,可以阻断翻译启动,从而干扰核糖体结合。在所提出的方法中,我们利用这种阻塞现象来量化限制性商业惯例与其共性和非共性绑定位点的绑定亲和力。为此,我们在报告器 mRNA 的起始区域插入一个测试绑定位点,并诱导测试 RBP 的表达。在RBP-RNA结合的情况下,我们观察到作为RBP浓度函数的对报告者表达的抑制。在绑定站点和 RBP 之间没有亲和力或非常低的亲和力的情况下,没有观察到明显的抑制。该方法在活细菌细胞中进行,不需要昂贵或精密的机械。它可用于量化和比较细菌中功能的不同限制性商业惯例与一组设计结合位点之间的结合亲和力。此方法可能不适合具有高结构复杂性的绑定站点。这是因为在没有RBP的情况下,复杂的mRNA结构可能抑制核糖体启动,这将导致基底报告者基因表达降低,因此对RBP结合的观察量较低。

所提出的方法利用RBP和核糖体之间的竞争,与mRNA分子结合(图1)。这种竞争反映在由于诱导剂浓度增加而降低mCherry水平,作为RBP-mCeran产量增加的函数。在增加mCerulean荧光的情况下,mCherry没有显著变化,可以推断出缺乏RBP结合。图 2描述了正应变和负应变的代表性结果。在图 2A中,OD、mCherry 和 mCerulean 通道在四个?...

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

定量细菌中蛋白质-RNA结合的测定

在这种方法中,我们使用在细菌细胞中的简单、实时的、报告学测定来量化RNA结合蛋白(RBPs)与共和结合位点的结合亲和力。测定基于对报告者基因的抑制。

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

由于RNA的复杂性质以及影响其与蛋白质相互作用的许多因素，体内蛋白质与RNA结合的特征仍然是一个重大挑战。RNA结合蛋白或RBP-RNA亲和力在活细菌细胞内测量。由于细胞环境同时影响RNA的结构以及由此产生的RBP-RNA结合，测量体内的亲和力对于理解自然系统中的这种相互作用至关重要。成功的秘诀是质粒的设计。该设计应使用几波增量，避免插入停止编子和帧移位突变。通过设计绑定站点盒式磁带来开始此协议。每个迷你基因包含一个EagI限制位点，40个碱基从五个基端的卡纳霉素抗性基因，pLac/Ara启动子，核糖体结合位点，AUG的mCherry基因，一个空间，一个RB结合位点，80个碱基从5个黄金端的mCherry基因，和一个阿帕利限制位点。为了提高测定的成功率，请为每个绑定站点设计三个绑定站点盒，并配有至少一个、两个和三个碱基的分空间。在执行消化的列纯化之前，使用EagI HF和PAPALI双消化迷你基因和目标载体。将消化的迷你基因装订到结合位骨干，其中包含其余mCherry报告基因、终止剂和卡那霉素抗性基因。然后将结扎溶液转化为大肠杆菌前10个细胞。通过桑格测序识别阳性转化剂后，在零下20摄氏度的96井格式中储存纯化质粒。还要将细菌菌株储存为甘油，在零下80摄氏度的96井格式中储存甘油。自定义顺序所需的 RBP 序列， 缺乏停止 codon 作为双链 DNA 迷你基因与限制位点在末端。将准备好的质粒转化为化学上称职的细菌细胞，这些细菌细胞已经含有RBP mCerulean质粒。为了节省时间，在含有相关抗生素的Luria-Bertani agar的8个通道板上使用8通道移液器对细胞进行镀盘。殖民地应在16小时内出现在37摄氏度。为每个双转化剂选择一个菌落，并转移至48个含有1.5毫升LB的井板，并服用适当的抗生素。在 37 摄氏度的温度下在 250 RPM 下生长 18 小时。将隔夜储存为零下80摄氏度的甘油库存，在96个井板中。早晨，程序机器人温暖180微升的半孔介质或SPM在96孔板。编程机器人准备诱导板。在干净的 96 孔板中，使用 SPM 准备由 95%BA 和 5%LB 组成的油井。井数对应于所需的诱导剂浓度数。将 C4-HSL 添加到诱导板中含有最高诱导剂浓度的孔中。接下来，对机器人进行编程，将每个最高浓度井中的介质连续稀释为 23 个浓度较低的介质，范围从零到 218 纳米摩尔。每种诱导剂稀释的体积应足以满足所有菌株。为了稀释隔夜菌株，将20微升细菌与180微升SPM混合在48个孔板中，使机器人稀释10倍。然后再次稀释10倍，将稀释溶液中的20微升放入适合荧光测量的预制应变板中。现在，程序机器人添加稀释诱导剂从诱导板到96井板与稀释应变根据最终浓度。在37摄氏度下摇动96个井板6小时。在此期间，每 30 分钟通过板读卡器测量 595 纳米的光密度或 OD 以及 mCherry 和 mCerulean 荧光。出于规范化目的，测量不添加单元格的 SPM 生长。此处显示为正应变的三维图。这些图描绘了原始OD水平、mCerulean荧光和mCherry荧光作为时间和诱导剂浓度的函数。mCerulean稳定状态表达水平计算为正应变和负应变的每个诱导剂浓度。还对正菌株和负菌株的每个诱导剂浓度计算了 mCherry 的生产成本。mCherry 的生产成本被绘制为平均 mCerulean 荧光平均超过两个菌株的两个生物重复的函数。对于表现出特定结合响应的正应变，将显示使用拟合公式的拟合 KRBP。对于负应变，未提取 KRBP 值。根据两个 10 个结合点在不同位置确定 30 种不同菌株的规范化剂量响应曲线。观察到三种类型的响应，高亲和力，低亲和力，没有亲和力。此处显示的两个 RBP 的定量 KRBP 结果，MS2 涂层蛋白和 PP7 涂层蛋白，以及 10 个不同的结合点盒。此处显示了 MS2 涂层蛋白的剂量响应曲线，该蛋白具有四个不同位置的突变结合位点，以演示突变间距对 mCherry 生产的影响。将显示测试的突变绑定站点的顺序。使用结构技术（如形状寻求）对蛋白质-RNA复合物的结构进行化学探测，并可视化哪些mRNA区域受RNA结合影响，这一点很重要。最近，我们以高吞吐量的方式使用此技术，同时测量 10，000 个绑定站点的亲和力。

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

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

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

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

量化腹部色素沉着<em&gt;黑腹果蝇</em

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

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

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

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

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

基于细菌选择和生长的盘状细胞基因工程

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

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...