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In vitro Transcription (IVT) and tRNA Binding Assay
体外转录(IVT)和tRNA结合试验   

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Abstract

This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The first part of the protocol also describes the in vitro transcription and labeling of the tRNA.

Keywords: T-box(T-box), EMSA(EMSA), RNA-RNA interaction(RNA-RNA相互作用), TRNA(tRNA), In vitro transcription(体外转录)

Materials and Reagents

  1. For in vitro transcription (IVT) in general
    1. T7 RNA polymerase including 5x T7 transcription buffer (Thermo Fisher Scientific, Fermentas, catalog number: EP0111 )
    2. RiboLock RNase inhibitor (Thermo Fisher Scientific, Fermentas, catalog number: EO0381 )
    3. 100 mM of ATP, UTP, GTP, CTP (NTP set) (Thermo Fisher Scientific, Fermentas, catalog number: R0481 )
    4. Recombinant DNase I (Life Technologies, Ambion®, catalog number: AM2235 )
    5. RNase-free water (Life Technologies, Ambion®, catalog number: AM9932 )
    6. [alpha 32P]-CTP (800 Ci/mmol, 10 µCi/µl) (PerkinElmer, catalog number: BLU008X )
    7. 1x T7 transcription buffer (see Recipes)
      Note: Templates will be generated by PCR, so all reagents for PCR are needed, too.

  2. For tRNA IVT in particular
    1. MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
    2. Guanosine 5’-monophosphate disodium salt hydrate from yeast (GMP) (as 100 mM stock solution in RNase-free water) (Sigma-Aldrich, catalog number: G8377 )
    3. Bio-Spin chromatography columns, Bio-Gel P-6 in Tris buffer (Bio-Rad Laboratories, catalog number: 732-6227 )
    4. OptiPhase Supermix liquid scintillation cocktail (PerkinElmer, catalog number: 1200-439 )
    5. Microbeta Starter kit plates (PerkinElmer, catalog number: 1450-486 )

  3. For denaturing Urea-PAGE
    1. Urea (Sigma-Aldrich, catalog number: U6504 )
    2. Rotiphorese 40% acrylamide/bis-acrylamide (19:1) solution (Carl Roth, catalog number: 3030.1 )
    3. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: 215589 ) [as 10% (w/v) solution in RNase-free water]
    4. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
    5. 2x MOPS buffer (e.g. Life Technologies, Ambion®, catalog number: AM9570 )
    6. Formamide (e.g. Life Technologies, Ambion®, catalog number: AM9342 )
    7. Formaldehyde (e.g. Sigma-Aldrich, catalog number: F8775 )
    8. Saccharose (e.g. Sigma-Aldrich, catalog number: S7903 )
    9. Bromophenol blue (e.g. Sigma-Aldrich, catalog number: B0126 )
    10. Xylencyanol (e.g. Sigma-Aldrich, catalog number: X4126 )
    11. 1x TBE buffer (see Recipes)
    12. 2x RNA loading dye (see Recipes)

  4. For native polyacrylamide gel electrophoresis (PAGE)
    1. 40% acrylamide/bis-acrylamide (19:1) solution, APS and TEMED as listed in C
    2. Glycerol (e.g. Sigma-Aldrich, catalog number: G5516 )
    3. Tris (e.g. Sigma-Aldrich, catalog number: T1503 )
    4. Boric acid (e.g. Sigma-Aldrich, catalog number: B7901 )
    5. EDTA (e.g. Sigma-Aldrich, catalog number: E9884 )
    6. 0.5x TBE buffer (see Recipes)
    7. 10x loading buffer (see Recipes)

Equipment

  1. Basic lab equipment for molecular biology with emphasis on RNase-free
    1. 0.5 ml RNase-free microfuge tubes (Applied Biosystems®, catalog number:  AM12300 )
    2. 1.5 ml RNase-free microfuge tubes (Applied Biosystems®, catalog number:  AM12400 )
    3. Microliter pipettes (e.g. Eppendorf) with tips and 15 ml or 50 ml tubes (e.g. SARSTEDT AG)
    4. Table-top centrifuge (e.g. Microcentrifuge, Eppendorf, catalog number:  5425 )
    5. Heating block for 1.5 ml and 0.5 ml tubes (e.g. Grant Instruments, Grant bio PCH-1 personal benchtop cooler/heaters)
      Note: Templates will be generated by PCR, so a standard PCR thermo cycler is needed, too.

  2. Lab equipment in designated area for radioactive work
    1. Liquid scintillation counter (PerkinElmer, model: 1450 LSC & Luminescence counter)
    2. Electrophoresis chamber, glass slides, spacers, clamps and combs for vertical PAGE (e.g. C.B.S. Scientific, catalog number: WSP2-SG-200 ), power supply (e.g. C.B.S. Scientific, catalog number: EPS-200-X)
    3. Plastic wrap, transparent sheets and Whatman paper (e.g. Sigma-Aldrich, catalog number: Z742422 )
    4. Gel dryer (Model 583 Gel Dryer) (Bio-Rad Laboratories, catalog number: 165-1746 ) with vacuum pump
    5. PhosphorImager (Fujifilm FLA-7000) (GE Healthcare, catalog number: 28-9558-09 ) and PhosphorImager screen (storage phosphor screens) (VWR International, catalog number: 28-9564 ) in cassette (VWR International, catalog number: 63-0035 )

Procedure

  1. In vitro transcription (IVT) of tRNAs with free 3’cca end
    1. Primer for amplification of the tRNA template from the bacterial chromosome by PCR need the T7 promoter sequence at their 5’ end for in vitro transcription by the T7 polymerase. For successful transcription, three purine bases, at best guanine, should follow the core promoter sequence (underlined): nnnnCTAATACGACTCACTATAGRRnnnnnn.… with the first G (in bold) as transcription start.
    2. PCR products for tRNA templates are cleaned up with the MinElute PCR Purification Kit according to the manufacturer’s protocol.
    3. The tRNA IVT reaction is set up in 20 µl volume as follows.
      1. Add 1x T7 reaction buffer, 20 U RiboLock™ RNase inhibitor, 0.5 mM ATP, GTP and UTP, 12 µM CTP, 9 mM GMP, 20 U T7 RNA polymerase and 2 µl of [alpha 32P]-CTP (800 Ci/mmol, 10 µCi/µl) to 4 pmol of DNA template (tRNA-PCR).
        Note: The reaction set up follows the protocol from Fermentas for Synthesis of Radiolabeled RNA Probes of High Specific Activity (Fermentas). The GMP is added to increase the 5’ monophosphorylation of the resulting tRNA molecules (according to Sampson and Uhlenbeck, 1988).
      2. Incubation of the reaction mix at 37 °C for <7 h to enrich transcription of short fragments and stop of reaction at -20 °C for 5 min.
      3. DNA template is removed by adding 1 U of recombinant DNase I and incubating at 37 °C for further 30 min.
      4. tRNA products have to be cleaned up using e.g. the Micro Bio-Spin™ chromatography columns with Bio-Gel P-6 in Tris buffer to ensure no small tRNA fragments are lost. Keep 1 µl of the reaction volume before the clean-up if you use the scintillation counter to measure the incorporation rate (see A4).
        Note: Other clean up kits are of course possible as long as loss of the desired product is avoided and unincorporated, radioactive nucleotides are removed. If more than one tRNA product can be detected by denaturing Urea-PAGE (see Figure 1), the correct band should rather be eluted from the gel. The recipe for denaturing Urea-PAGE can be found after this section.
    4. To establish the incorporation rate of radionucleotides during IVT into the produced tRNA, 1 µl of the reaction volume before and after clean-up is used for scintillation count.
      1. OptiPhase Supermix liquid scintillation cocktail (50 µl per well) are pipetted into a rigid 96-well plate and the 1 µl samples are added, with one empty well between samples to avoid cross-counting of wells.
      2. In parallel, a dilution series of the utilized radionucleotide [alpha 32P]-CTP is added to the same plate to obtain a standard curve for the cpm (counts per min).
      3. The plate is sealed and placed in the liquid scintillation counter. The cpm from the standard curve will give the cpm value per µCi. This will change with each experiment, due to the half life of P32. The cpm values of the sample before and after clean-up helps to calculate the incorporation rate of radionucleotides and subsequently the RNA yield after IVT to determine the molarity of tRNA to be used in the binding assay.
        Note: Incorporation rate and RNA yield can be calculated with help of an article by Ambion (http://www.lifetechnologies.com/de/de/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/determining-rna-probe-specific-activity-and-yield.html). If no scintillation counter is available, incorporation rate can also be estimated through Geiger counter values obtained before and after clean-up and the RNA yield and molarity calculated from there.

  2. Recipe for denaturing Urea-PAGE
    1. A 10% (w/v) acrylamide/ 8 M urea gel was chosen for the separation of 78 nt tRNA molecules on a small size gel (10 x 10 cm).
      1. 3.36 g urea are solved in 1x TBE (up to volume of 5.25 ml) by stirring for > 4 h at room temperature. 1.75 ml 40% acrylamide/bis-acrylamide (19: 1) solution are added to the 8 M urea solution to reach 7 ml total volume.
        Note: The total volume depends on size of glass plates and thickness of spacers used. Here we work with 10 x 10 cm plates and 1 mm thickness and need 7 ml volume. The urea-acrylamide mix can be prepared in advance in a bigger volume and stored at 4 °C.
      2. Clean glass slides of desired size are set up with spacer on left and right and either put in a gel casting chamber or fixed with clamps on both sides.
      3. 35 μl [0.5% (v/v)] of 10% APS and 15 μl 0.2% (v/v) TEMED are added to the urea-acrylamide mix to catalyze the polymerization process.
      4. The mix is quickly poured between the glass slides (e.g. with 10 ml glass pipette) and the comb with desired amount of wells adjusted in the gel. Avoid air bubbles!
    2. The fully polymerized gel was pre-run in 1x TBE buffer at 250 V at room temperature for 40 min. Remove the comb for that.
    3. tRNA samples were mixed with 2x RNA loading dye and heat denatured at 85 °C for 5 min before loading.
      Note:  Rinse the wells thoroughly right before loading the samples!
    4. Electrophoresis settings are 250 V at room temperature to run for 1 h in 1x TBE buffer.
    5. After electrophoresis run the gel is transferred to Whatman paper and covered in a transparent sheet before exposure of a PhosphorImager screen (couple of hours is usually sufficient), followed by scan with the PhosphorImager FLA-7000 (see Figure 1).
      Note: Do not dry Urea-PA gels with the gel dryer, as they will disintegrate.

  3. IVT-RNA-tRNA binding assay
    1. A typical 10 μl reaction volume contains all components for T7 in vitro transcription:
      1. 1x T7 transcription buffer, 10 U of RiboLock RNase inhibitor, 6 U of T7 RNA polymerase and 0.5 mM NTPs (ATP, UTP, GTP, CTP). DNA template (PCR product) for the leader RNA is added to 8 nM and the radiolabeled tRNA to 50 nM.
        Note: The leader RNA template has to contain a T7 promoter sequence at the 5’ end as described in A1.
      2. Control reactions are set up accordingly without tRNA but with 12 µM CTP and 1 µl [alpha 32P]-CTP (800 Ci/mmol, 10 µCi /µl) to check for IVT efficiency.
      3. The reaction mix is incubated at 37 °C for 2 h, which is sufficient for the 440 nt transcript.
      4. 2 µl of 10x loading buffer are added and the samples immediately loaded onto a non-denaturing polyacrylamide gel.
    2. A 6% (w/v) acrylamide gel was chosen to separate a 78 nt tRNA from the complex with a ~440 nt RNA on a medium size vertical gel.
      1. Clean glass slides of desired size are set up with spacer on left and right and either put in a gel casting chamber or fixed with clamps on both sides.
      2. 1.5 ml 40% acrylamide/bis-acrylamide (19:1) solution and 0.5 ml 10x TBE are mixed with RNase-free water to 10 ml volume and then 40 μl [0.4% (v/v)] of 10% APS and 20 μl [0.2% (v/v)] TEMED are added to catalyze the polymerization process.
        Note: The total volume depends on size of glass plates and thickness of spacers used. Here we work with 10 x 15 cm plates and 1 mm thickness and need 10 ml volume. Higher percentage of TEMED than usual was used to achieve a very quick polymerization.
      3. The mix is quickly poured between the glass slides (e.g. with 10 ml glass pipette) and the comb with desired amount of wells adjusted in the gel. Avoid air bubbles!
      4. The fully polymerized gel has to be pre-run in 0.5x TBE buffer at 200 V at 4 °C for at least 40 min. Remove the comb for that. Rinse the wells before loading samples.
      5. Immediately load the samples after binding reaction.
        Note: If possible, load the samples quickly while electrophoresis chamber is already running to avoid disassociation of the complex.
      6. Electrophoresis settings are 100 V (= 6.7 V/cm), 5 W at 4 °C to run for >3 h.
        Note: These settings depend mainly on the size difference between unbound and bound labeled RNA moiety and on the size of the gel used (so it should not run out the bottom but be clearly separated). It is much better to cool the gel while running (so either in a 4 °C room or with a buffer cooling system).
    3. After electrophoresis, the gel is transferred onto a Whatman paper and covered with plastic wrap, then dried by vacuum gel dryer at 80 °C for < 30 min.
    4. Detection of the radiolabeled RNA complex is achieved by exposure of a PhosphorImager screen (often over night), followed by scan with the PhosphorImager FLA-7000 (see Figure 2).

Representative data



Figure 1. Scanned image of denaturing Urea-PAGE of labelled tRNAs. This example shows dominant single products for most tRNA IVT reactions (arrow), but also a diffuse pattern of several products in one sample (lane 5). This sample would result in an unspecific binding reaction, thus the IVT must be repeated or the correct band eluted from the gel. lane 1: size marker; lane 2 - 6: different in vitro transcribed tRNAs.


Figure 2. Scanned image of native PAGE gel after binding assay. This is an example of the separation of unbound, small tRNA* at the bottom of the gel and the tRNA* bound to the much bigger T-box RNA (arrow), which was transcribed in vitro in the presence of pre-formed, radiolabelled(*) tRNA. lane 1: control IVT reaction of T-box RNA* without tRNA; lane 2: T-box RNA IVT with tRNA*fMet; lane 3: T-box RNA IVT with tRNA*Cys (not binding to T-box RNA).

Notes

  1. The most variable component in our hands was the in vitro transcription of the tRNA molecules. Only if one dominant tRNA product was achieved [controlled by denaturing PAGE with 10 % (w/v) acrylamide/ 8 M urea, see Figure 1], then the detected binding interaction was strong. If one discovers a high diversity in the produced tRNA moiety, one might be better advised to elute the labelled tRNA band from the urea-acrylamide gel before the binding assay.

Recipes

  1. 5x T7 transcription buffer (usually supplied with T7 polymerase)
    200 mM Tris/HCl (pH 7.9)
    50 mM DTT
    30 mM MgCl2
    50 mM NaCl
    10 mM spermidine
  2. 10x TBE buffer (1 L)
    0.89 M Tris
    0.9 M boric acid
    25 mM EDTA
    Solve 108 g Tris and 55 g boric acid in 950 ml distilled water
    Add 50 ml 0.5 M EDTA (pH 8.0)
  3. 2x RNA loading dye
    2x MOPS buffer
    65% (v/v) formamide
    4.4% (v/v) formaldehyde
    2% (w/v) saccharose
    0.1% (w/v) bromophenol blue
    0.1% (w/v) xylencyanol
  4. 10x loading buffer
    40% (v/v) glycerol
    0.25x TBE

Acknowledgments

This work was funded by the German Research Foundation (DFG), Transregio34 “Pathophysiology of staphylococci in the post-genomic era”, and my PhD stipend was granted by the European Social Fund (ESF). The protocol presented here was developed in the lab of PD Dr. Wilma Ziebuhr during my research time at the Queen’s University Belfast (QUB).

References

  1. Fermentas T7 IVT protocols: http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/t7-rna-polymerase/
  2. Sampson, J. R. and Uhlenbeck, O. C. (1988). Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci U S A 85(4): 1033-1037.
  3. Schoenfelder, S. M., Marincola, G., Geiger, T., Goerke, C., Wolz, C. and Ziebuhr, W. (2013). Methionine biosynthesis in Staphylococcus aureus is tightly controlled by a hierarchical network involving an initiator tRNA-specific T-box riboswitch. PLoS Pathog 9(9): e1003606.

简介

该方案描述了(i)"活"体外RNA转录与(ii)通过放射性标记的预先形成的tRNA的结合,然后是天然凝胶电泳和磷光成像仪扫描以显现复合物的偶联。 必要性来自一种RNA在不存在其相互作用配偶体时形成的稳定结构。 T盒前导RNA,转录控制系统,折叠成热力学非常稳定的茎 - 环结构,没有tRNA存在,这使得体外结合两个预先形成的RNA的相互作用非常困难。 因此,我调整结合测定以模拟细菌细胞中的"天然"情况,其中预先形成的稳定的tRNA已经存在,而T盒前导RNA被RNA聚合酶主动转录。 方案的第一部分还描述了体外转录和tRNA的标记。

关键字:T-box, EMSA, RNA-RNA相互作用, tRNA, 体外转录

材料和试剂

  1. 对于体外转录(IVT)
    1. 包括5×T7转录缓冲液(Thermo Fisher Scientific,Fermentas,目录号:EP0111)的T7 RNA聚合酶
    2. RiboLock RNA酶抑制剂(Thermo Fisher Scientific,Fermentas,目录号:EO0381)
    3. 100mM ATP,UTP,GTP,CTP(NTP组)(Thermo Fisher Scientific,Fermentas,目录号:R0481)
    4. 重组DNA酶I(Life Technologies,Ambion ,目录号:AM2235)
    5. 无RNase的水(Life Technologies,Ambion ,目录号:AM9932)
    6. (PercelElmer,目录号:BLU008X)
      PUP(800Ci/mmol,10μCi/
    7. 1x T7转录缓冲液(参见配方)
      注意:模板将通过PCR生成,因此也需要PCR的所有试剂。

  2. 特别是对于tRNA IVT
    1. MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
    2. 来自酵母的鸟苷5'-单磷酸二钠盐水合物(GMP)(作为100mM在无RNA酶的水中的储备溶液)(Sigma-Aldrich,目录号:G8377)
    3. Bio-Spin层析柱,Tris缓冲液中的Bio-Gel P-6(Bio-Rad Laboratories,目录号:732-6227)
    4. OptiPhase Supermix液体闪烁混合物(PerkinElmer,目录号:1200-439)
    5. Microbeta入门试剂盒平板(PerkinElmer,目录号:1450-486)

  3. 用于变性尿素PAGE
    1. 脲(Sigma-Aldrich,目录号:U6504)
    2. Rotiphorese 40%丙烯酰胺/双丙烯酰胺(19:1)溶液(Carl Roth,目录号:3030.1)
    3. 过硫酸铵(APS)(Sigma-Aldrich,目录号:215589)[作为在无RNA酶的水中的10%(w/v)溶液]
    4. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
    5. 2x MOPS缓冲液(例如Life Technologies,Ambion ,目录号:AM9570)
    6. 甲酰胺(例如Life Technologies,Ambion ®,目录号:AM9342)
    7. 甲醛(例如Sigma-Aldrich,目录号:F8775)
    8. 蔗糖(例如Sigma-Aldrich,目录号:S7903)
    9. 溴酚蓝(例如Sigma-Aldrich,目录号:B0126)
    10. 二甲醇(例如Sigma-Aldrich,目录号:X4126)
    11. 1x TBE缓冲区(参见配方)
    12. 2x RNA加载染料(参见配方)

  4. 对于天然聚丙烯酰胺凝胶电泳(PAGE)
    1. 40%丙烯酰胺/双丙烯酰胺(19:1)溶液,APS和TEMED,如C所列
    2. 甘油(例如Sigma-Aldrich,目录号:G5516)
    3. Tris(例如Sigma-Aldrich,目录号:T1503)
    4. 硼酸(例如Sigma-Aldrich,目录号:B7901)
    5. EDTA(例如Sigma-Aldrich,目录号:E9884)
    6. 0.5x TBE缓冲区(参见配方)
    7. 10x加载缓冲区(参见配方)

设备

  1. 分子生物学的基本实验室设备,强调无核糖核酸酶
    1. 0.5ml无RNase的微量离心管(Applied Biosystems ,目录号:AM12300)
    2. 1.5ml无RNase的微量离心管(Applied Biosystems ,目录号:AM12400)
    3. 具有尖端和15ml或50ml管(例如SARSTEDT AG)的微量移液管(例如Eppendorf)
    4. 台式离心机(例如Microcentrifuge,Eppendorf,目录号:5425)
    5. 用于1.5ml和0.5ml管(例如Grant Instruments,Grant bio PCH-1个人台式冷却器/加热器)的加热块
      注意:模板将通过PCR生成,因此也需要标准的PCR热循环仪。

  2. 实验室设备在指定区域进行放射性工作
    1. 液体闪烁计数器(PerkinElmer,型号:1450LSC&发光计数器)
    2. 用于垂直PAGE(例如CBS Scientific,目录号:WSP2-SG-200),电源(例如CBS Scientific,Inc。)的电泳室,载玻片,间隔物,夹具和梳子 目录号:EPS-200-X)
    3. 塑料包装,透明片材和Whatman纸(例如,Sigma-Aldrich,目录号:Z742422)
    4. 凝胶干燥器(583型凝胶干燥器)(Bio-Rad Laboratories,目录号:165-1746)用真空泵
    5. 在盒(VWR International,目录号:63-92)中的荧光成像器(Fujifilm FLA-7000)(GE Healthcare,目录号:28-9558-09)和PhosphorImager屏(存储荧光屏)(VWR International,目录号:28-9564) 0035)

程序

  1. 具有游离3'cca末端的tRNA的体外转录(IVT)
    1. 用于通过PCR扩增来自细菌染色体的tRNA模板的引物需要T7启动子序列在它们的5'末端用于通过T7聚合酶的体外转录。为了成功转录,三个嘌呤碱基,最好是鸟嘌呤,应该跟随核心启动子序列(下划线):nnnnCTAATACGACTCACTATAGRRnnnnnn ...第一个G(以粗体表示)作为转录起始。
    2. 用于tRNA模板的PCR产物根据制造商的方案用MinElute PCR纯化试剂盒净化
    3. tRNA IVT反应如下在20μl体积中建立。
      1. 加入1×T7反应缓冲液,20U RiboLock TM RNA酶抑制剂,0.5mM ATP,GTP和UTP,12μMCTP,9mM GMP,20U T7 RNA聚合酶和2μl的α32 P] -CTP(800Ci/mmol,10μCi/μ 1)至4pmol DNA模板(tRNA-PCR)。
        注意:反应设置遵循来自Fermentas for Synthesis of Radiolabeled RNA Probes of High Specific Activity(Fermentas)的方案。添加GMP以增加所得tRNA分子的5'单磷酸化(根据Sampson和Uhlenbeck,1988)。
      2. 在37℃下孵育反应混合物<7小时以富集短片段的转录并在-20℃下停止反应5分钟。
      3. 通过加入1U重组DNA酶I并在37℃下再温育30分钟除去DNA模板
      4. tRNA产物必须使用例如Micro Bio-Spin TM层析柱用Bio-Gel P-6在Tris缓冲液中清洗以确保没有小的tRNA片段丢失。如果使用闪烁计数器测量掺入率,则在清理前保留1μl反应体积(见A4)。
        注意:其他清洁试剂盒当然是可能的,只要所需产物的损失被避免和未掺入,放射性核苷酸被去除。如果通过变性尿素PAGE可以检测多于一种tRNA产物(参见图1),则应该从凝胶洗脱正确的条带。可以在本节后找到变性尿素PAGE的配方。
    4. 为了建立放射性核苷酸在IVT期间掺入所产生的tRNA中的掺入率,将1μl在清除前后的反应体积用于闪烁计数。
      1. 将OptiPhase Supermix液体闪烁混合物(每孔50μl)移液到刚性96孔板中,并加入1μl样品,样品之间有一个空孔,以避免孔的交叉计数。
      2. 平行地,将使用的放射性核苷酸[α32 P] -CTP的稀释系列加入到同一板中以获得cpm(每分钟计数)的标准曲线。
      3. 将板密封并置于液体闪烁计数器中。来自标准曲线的cpm将给出每μCi的cpm值。由于P 32的半衰期,这将随着每个实验而改变。清除之前和之后的样品的cpm值有助于计算放射性核苷酸的掺入率,并且随后计算IVT后的RNA产量,以确定在结合测定中使用的tRNA的摩尔浓度。
        注意:可以通过Ambion的文章( http://www.lifetechnologies.com/de/de /home/references/ambion-tech-support/nuclease-enzymes/tech-notes/determining-rna-probe-specific-activity-and-yield.html )。如果没有闪烁计数器,也可以通过在清除之前和之后获得的盖革计数器值以及从其计算的RNA产率和摩尔浓度来估计掺入率。

  2. 用于变性尿素PAGE的配方
    1. 选择10%(w/v)丙烯酰胺/8M尿素凝胶用于在小尺寸凝胶(10×10cm)上分离78nt的tRNA分子。
      1. 将3.36g脲溶解在1×TBE(至体积为5.25ml)中,通过搅拌> 在室温下4小时。 向8M尿素溶液中加入1.75ml 40%丙烯酰胺/双丙烯酰胺(19:1)溶液,使总体积达到7ml。
        注意:总体积取决于玻璃板的尺寸和使用的垫片的厚度。 这里我们使用10×10厘米板和1毫米厚度,需要7毫升的体积。 脲 - 丙烯酰胺混合物可以预先在更大的体积中制备并在4℃下储存。
      2. 具有所需尺寸的清洁玻璃载玻片在左侧和右侧设置有间隔物,并且放置在凝胶浇铸室中或者在两侧用夹具固定。
      3. 向脲 - 丙烯酰胺混合物中加入35μl[0.5%(v/v)] 10%APS和15μl0.2%(v/v)TEMED以催化聚合过程。
      4. 将混合物快速倒入载玻片(例如用10ml玻璃移液管)和梳子之间,并在凝胶中调节所需量的孔。避免气泡!
    2. 将完全聚合的凝胶在1×TBE缓冲液中在250V在室温下预运行40分钟。取下梳子。
    3. 将tRNA样品与2x RNA加载染料混合,并在加载前在85℃热变性5分钟。
      注意:在装载样品之前,立即冲洗孔!
    4. 电泳设置为在室温下250V,在1x TBE缓冲液中运行1小时。
    5. 电泳后,将凝胶转移到Whatman纸上,并在PhosphorImager筛网曝光(几小时通常足够)之前覆盖在透明片材上,然后扫描 与PhosphorImager FLA-7000(参见图1)。
      注意:不要用凝胶干燥器干燥尿素-PA凝胶,因为它们会崩解。

  3. IVT-RNA-tRNA结合测定
    1. 典型的10μl反应体积包含T7体外转录的所有组分:
      1. 1x T7转录缓冲液,10U RiboLock RNA酶抑制剂,6U T7 RNA聚合酶和0.5mM NTP(ATP,UTP,GTP,CTP)。 将用于前导RNA的DNA模板(PCR产物)添加至8nM,并将放射性标记的tRNA添加至50nM。
        注意:前导RNA模板必须在A1的5'末端含有T7启动子序列。
      2. 因此,没有tRNA而是使用12μMCTP和1μl[α32 P] -CTP(800Ci/mmol,10μCi/μl)来设置对照反应以检查IVT效率。
      3. 将反应混合物在37℃下孵育2小时,这对于440nt的转录物是足够的。
      4. 加入2μl10×上样缓冲液,将样品立即装入非变性聚丙烯酰胺凝胶上
    2. 选择6%(w/v)丙烯酰胺凝胶以在中等大小的垂直凝胶上用〜440nt的RNA从复合物中分离78nt的tRNA。
      1. 具有所需尺寸的清洁玻璃载玻片在左侧和右侧设置有间隔物,并且放置在凝胶浇铸室中或者在两​​侧用夹具固定。
      2. 将1.5ml 40%丙烯酰胺/双丙烯酰胺(19:1)溶液和0.5ml 10×TBE与无RNA酶的水混合至10ml体积,然后加入40μl[0.4%(v/v)] 10%APS和20μl μl[0.2%(v/v)] TEMED以催化聚合过程。
        注意:总体积取决于玻璃板的尺寸和使用的垫片的厚度。这里我们使用10 x 15厘米板和1毫米厚度,需要10毫升的体积。使用比通常更高的TEMED百分比来实现非常 快速聚合。
      3. 将混合物快速倒入载玻片(例如用10ml玻璃移液管)和梳子之间,并在凝胶中调节所需量的孔。避免气泡!
      4. 完全聚合的凝胶必须在0.5×TBE缓冲液中在200V在4℃预运行至少40分钟。取下梳子。在装样前冲洗孔。
      5. 在结合反应后立即装载样品。
        注意:如果可能,请在电泳室已经运行时快速装载样品,以避免复合体分离。
      6. 电泳设置为100V(= 6.7V/cm),5W在4℃运行> 3小时。
        注意:这些设置主要取决于未结合的和结合的标记的RNA部分之间的大小差异以及所使用的凝胶的大小(因此它不应当在底部,而是应当清楚地分开)。在运行时更好地冷却凝胶(因此在4℃的房间或缓冲冷却系统中)。
    3. 电泳后,将凝胶转移到Whatman纸上,用塑料包裹物覆盖,然后通过真空凝胶干燥器在80℃下干燥, 30分钟。
    4. 放射性标记的RNA复合物的检测通过曝光PhosphorImager筛(通常过夜),然后用PhosphorImager FLA-7000(参见图2)进行扫描来实现。

代表数据



图1.标记的tRNA的变性尿素PAGE的扫描图像。该实施例显示大多数tRNA IVT反应的主要单一产物(箭头),而且一个样品中几种产物的扩散模式(泳道5 )。该样品将导致非特异性结合反应,因此IVT必须重复或正确的条带从凝胶中洗脱。泳道1:大小标记;泳道2-6:不同的体外转录的tRNA

图2.结合测定后的原生PAGE凝胶的扫描图像这是在凝胶底部分离未结合的小tRNA *的实例,并且tRNA *与更大的T- (箭头),其在预形成的放射性标记的(*)tRNA的存在下在体外转录。泳道1:控制T-box RNA *无tRNA的IVT反应;泳道2:具有tRNA * fMet的T盒RNA IVT;泳道3:具有tRNA * Cys(不结合T盒RNA)的T盒RNA IVT。

笔记

  1. 我们手中最可变的组分是tRNA分子的体外转录。只有获得一个显性tRNA产物(通过用10%(w/v)丙烯酰胺/8M尿素的变性PAGE控制,参见图1],然后检测到的结合相互作用强。如果发现产生的tRNA部分的高度多样性,则可以更好地建议在结合测定前从脲 - 丙烯酰胺凝胶洗脱标记的tRNA条带。

食谱

  1. 5x T7转录缓冲液(通常与T7聚合酶一起提供) 200mM Tris/HCl(pH7.9)
    50 mM DTT
    30mM MgCl 2/v/v 50mM NaCl 10mM亚精胺
  2. 10x TBE缓冲液(1 L)
    0.89M Tris
    0.9M硼酸 25mM EDTA
    溶解108克Tris和55克硼酸在950毫升蒸馏水中的溶液 加入50ml 0.5M EDTA(pH8.0)
  3. 2x RNA上样染料
    2x MOPS缓冲区
    65%(v/v)甲酰胺 4.4%(v/v)的甲醛 2%(w/v)蔗糖 0.1%(w/v)溴酚蓝
    0.1%(w/v)二甲苯甲醇
  4. 10x加载缓冲区
    40%(v/v)甘油 0.25x TBE

致谢

这项工作由德国研究基金会(DFG),Transregio34"后基因组时代的葡萄球菌的病理生理学"资助,我的博士津贴是由欧洲社会基金(ESF)授予的。 这里提出的协议是在博士Wilma Ziebuhr实验室在我的研究时间在贝尔法斯特女王大学(QUB)。

参考文献

  1. Fermentas T7 IVT协议: http://www.thermoscientificbio .com/dna-and-rna修饰酶/t7-rna-polymerase/
  2. Sampson,J.R。和Uhlenbeck,O.C。(1988)。 体外转录的未修饰的酵母苯丙氨酸转移RNA的生物化学和物理表征 。 Proc Natl Acad Sci USA 85(4):1033-1037。
  3. Schoenfelder,S. M.,Marincola,G.,Geiger,T.,Goerke,C.,Wolz,C.and Ziebuhr,W。 葡萄球菌中的甲硫氨酸生物合成aureus 受到包含启动子tRNA特异性T盒核糖开关的分层网络的严格控制。

    9(9):e1003606。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Schoenfelder, S. M. (2014). In vitro Transcription (IVT) and tRNA Binding Assay. Bio-protocol 4(18): e1234. DOI: 10.21769/BioProtoc.1234.
  2. Schoenfelder, S. M., Marincola, G., Geiger, T., Goerke, C., Wolz, C. and Ziebuhr, W. (2013). Methionine biosynthesis in Staphylococcus aureus is tightly controlled by a hierarchical network involving an initiator tRNA-specific T-box riboswitch. PLoS Pathog 9(9): e1003606.
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