JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
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Acknowledgements
Materials
Referências
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Genética

定量细菌中蛋白质-RNA结合的测定

Published: June 12, 2019
doi:
10.3791/59611
, , , ,
1Department of Biotechnology and Food Engineering,Technion-Israel Institute of Technology, 2Russell Berrie Nanotechnology Institute,Technion-Israel Institute of Technology

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 系统准备 结合位点质粒的设计 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、…

Representative Results

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?…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

  1. Cerretti, D. P., Mattheakis, L. C., Kearney, K. R., Vu, L., Nomura, M. Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein. Journal of Molecular Biology. 204 (2), 309-329 (1988).
  2. Babitzke, P., Baker, C. S., Romeo, T. Regulation of translation initiation by RNA binding proteins. Annual Review of Microbiology. 63, 27-44 (2009).
  3. Van Assche, E., Van Puyvelde, S., Vanderleyden, J., Steenackers, H. P. RNA-binding proteins involved in post-transcriptional regulation in bacteria. Frontiers in Microbiology. 6, 141 (2015).
  4. Chappell, J., Watters, K. E., Takahashi, M. K., Lucks, J. B. A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future. Current Opinion in Chemical Biology. 28, 47-56 (2015).
  5. Wagner, T. E., et al. Small-molecule-based regulation of RNA-delivered circuits in mammalian cells. Nature Chemical Biology. 14 (11), 1043 (2018).
  6. Bendak, K., et al. A rapid method for assessing the RNA-binding potential of a protein. Nucleic Acids Research. 40 (14), e105 (2012).
  7. Strein, C., Alleaume, A. -. M., Rothbauer, U., Hentze, M. W., Castello, A. A versatile assay for RNA-binding proteins in living cells. RNA. 20 (5), 721-731 (2014).
  8. Ule, J., Jensen, K. B., Ruggiu, M., Mele, A., Ule, A., Darnell, R. B. CLIP identifies Nova-regulated RNA networks in the brain. Science. 302 (5648), 1212-1215 (2003).
  9. Lee, F. C. Y., Ule, J. Advances in CLIP Technologies for Studies of Protein-RNA Interactions. Molecular Cell. 69 (3), 354-369 (2018).
  10. Katz, N., et al. An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene. ACS Synthetic Biology. 7 (12), 2765-2774 (2018).
  11. Watters, K. E., Yu, A. M., Strobel, E. J., Settle, A. H., Lucks, J. B. Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Methods. 103, 34-48 (2016).
  12. Saito, H., et al. Synthetic translational regulation by an L7Ae-kink-turn RNP switch. Nature Chemical Biology. 6 (1), 71-78 (2010).
  13. Gott, J. M., Wilhelm, L. J., Uhlenbeck, O. C. RNA binding properties of the coat protein from bacteriophage GA. Nucleic Acids Research. 19 (23), 6499-6503 (1991).
  14. Peabody, D. S. The RNA binding site of bacteriophage MS2 coat protein. The EMBO Journal. 12 (2), 595-600 (1993).
  15. Lim, F., Peabody, D. S. RNA recognition site of PP7 coat protein. Nucleic Acids Research. 30 (19), 4138-4144 (2002).
  16. Lim, F., Spingola, M., Peabody, D. S. The RNA-binding Site of Bacteriophage Qβ Coat Protein. Journal of Biological Chemistry. 271 (50), 31839-31845 (1996).
  17. Gibson, D. G., et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 6 (5), 343-345 (2009).
  18. . Optimizing Restriction Endonuclease Reactions Available from: https://international.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions (2018)
  19. . Wizard® SV Gel and PCR Clean-Up System Protocol Available from: https://worldwide.promega.com/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/ (2018)
  20. . Ligation Protocol with T4 DNA Ligase (M0202) Available from: https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202 (2018)
  21. . Routine Cloning Using Top10 Competent Cells – US Available from: https://www.thermofisher.com/us/en/home/references/protocols/cloning/competent-cells-protocol/routine-cloning-using-top10-competent-cells.html (2018)
  22. . NucleoSpin Plasmid – plasmid Miniprep kit Available from: https://www.mn-net.com/ProductsBioanalysis/DNAandRNApurification/PlasmidDNApurificationeasyfastreliable/NucleoSpinPlasmidplasmidMiniprepkit/tabid/1379/language/en-US/Default.aspx (2018)
  23. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., Roe, B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. Journal of Molecular Biology. 143 (2), 161-178 (1980).
  24. . Protocol – How to Create a Bacterial Glycerol Stock Available from: https://www.addgene.org/protocols/create-glycerol-stock/ (2018)
  25. . Making your own chemically competent cells Available from: https://international.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells (2018)
  26. . Luria-Bertani (LB) Medium Preparation · Benchling Available from: https://benchling.com/protocols/gdD7XI0J/luria-bertani-lb-medium-preparation (2018)
  27. Delebecque, C. J., Silver, P. A., Lindner, A. B. Designing and using RNA scaffolds to assemble proteins in vivo. Nature Protocols. 7 (10), 1797-1807 (2012).
  28. Hocine, S., Raymond, P., Zenklusen, D., Chao, J. A., Singer, R. H. Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nature Methods. 10 (2), 119-121 (2013).
  29. Espah Borujeni, A., et al. Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences. Nucleic Acids Research. 45 (9), 5437-5448 (2017).
  30. Ding, Y., et al. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature. 505, (2013).
  31. Rouskin, S., Zubradt, M., Washietl, S., Kellis, M., Weissman, J. S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature. 505 (7485), 701-705 (2014).
  32. Lucks, J. B., et al. Multiplexed RNA structure characterization with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proceedings of the National Academy of Sciences of the United States of America. 108 (27), 11063-11068 (2011).
  33. Spitale, R. C., et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 519 (7544), 486 (2015).
  34. Watters, K. E., Abbott, T. R., Lucks, J. B. Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq. Nucleic Acids Research. 44 (2), e12 (2016).
  35. Flynn, R. A., et al. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature Protocols. 11 (2), 273-290 (2016).
  36. Bernardi, A., Spahr, P. -. F. Nucleotide Sequence at the Binding Site for Coat Protein on RNA of Bacteriophage R17. Proceedings of the National Academy of Sciences of the United States of America. 69 (10), 3033-3037 (1972).

Tags

Protein-RNA BindingRNA Binding ProteinRBP-RNA AffinityIn VivoPlasmid DesignBinding Site CassetteMCherry Reporter GeneRBP SequenceBacterial TransformationGlycerol Stocks

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Katz, N., Cohen, R., Atar, O., Goldberg, S., Amit, R. An Assay for Quantifying Protein-RNA Binding in Bacteria. J. Vis. Exp. (148), e59611, doi:10.3791/59611 (2019).
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