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Oct 2019

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Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
α-变形杆菌核糖体的纯化及Northern印迹分析   

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Abstract

Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris. Following this protocol, RNA can be extracted from either purified ribosomes or directly from cell cultures, and ribosomal RNAs quantified using Northern blot. This protocol gives an example of studying ribosomes in a bacterium other than the commonly used E. coli. The challenge of performing Northern blots with rRNA is also addressed in detail.

Keywords: Ribosome profile (Ribosome profile), capillary transfer (毛细管转移), rRNA (核糖体RNA), Northern blot (Northern印迹), Rhodopseudomonas palustris (沼泽红假单胞菌)

Background

The fate of bacterial cells is closely linked to their ribosomes. Our recent study showed that active ribosomes play an important role in the survival mechanisms of nutrient-deprived R. palustris cells (Yin et al., 2019). The ribosomes are purified via a series of ultracentrifugations with a protocol optimized from the classical methods (Lawrence et al., 2016). Ribosomal RNA populations are detected with Northern blot employing a less frequently used capillary transfer system. The state of ribosomes can be greatly affected by the details of purification steps. These methods, described here in detail, should be of broad interest to researchers who study the translation apparatus in a wide variety of bacteria.

Materials and Reagents

Note: All reagents can be made de novo or purchased from different vendors as long as they are RNase free.

  1. Centrifuge tubes, RNase-free (ThermoFisher AM12400 or equivalent). Use ultracentrifuge compatible tube for ultracentrifugation (please refer to the manual of reader’s ultracentrifuge for more information).

  2. Screw-cap tube, 2 ml (Sigma-Aldrich, BR780758 or equivalent)

  3. Costar Sterile Disposable Reagent Reservoirs (Fisher Scientific, catalog number: 07-200-128)

  4. HEPES-KOH, 1 M, pH 7.5 (Boston Bioproduct, catalog number: BBH-75-K)

  5. MgCl2, 1 M (Boston Bioproduct, catalog number: BM-670)

  6. NaCl, Molecular Biology Grade (Promega Corp, catalog number: H5271)

  7. Recombinant RNasin Ribonuclease Inhibitor (Promega Corp, catalog number: N2511)

  8. miRNeasy Mini Kit (Qiagen, catalog number: 217004)

  9. Agarose, molecular biology grade

  10. UltraPure DNase/RNase-Free Distilled Water (Invitrogen, catalog number: 10977-023)

  11. Amersham Hybond-XL membrane (GE Life Sciences)

  12. 10x Tris-borate-EDTA (TBE) buffer (Thermo Fisher Scientific, catalog number: AM9863)

  13. 20x SSC buffer (Sigma-Aldrich, catalog number: S6639)

  14. DIG RNA Labeling Mix (Sigma-Aldrich, catalog number: 11277073910)

  15. DIG Easy Hyb (Sigma-Aldrich, catalog number: 11603558001)

  16. DIG Wash and Block Buffer Set (Sigma-Aldrich, catalog number: 11585762001)

  17. Anti-Digoxigenin-AP, Fab fragments (Sigma-Aldrich, catalog number: 11093274910)

  18. CDP-Star (Sigma-Aldrich, catalog number: 11685627001)

  19. RNA Gel Loading Dye (2x) (ThermoFisher, catalog number: R0641)

  20. Buffer A1 (10x) (see Recipes)

  21. Lysis buffer (see Recipes)

Equipment

Note: The working bench as well as electrophoresis, transfer and Northern blot systems should be cleaned with RNaseZap or equivalent to decontaminate RNase before use. Items below that do not have a catalog number listed can be obtained from any reliable supplier.

  1. Mini-Beadbeater-24 (Bio Spec Products Inc., catalog number: 112011)

  2. Zirconia/Silica Beads, 0.1 mm dia (Bio Spec Products Inc., catalog number: 11079101z)

  3. Beckman TL-1000 ultracentrifuge (discontinued model, can be substituted with equivalent ultracentrifuge with temperature-control). Please make sure to use compatible centrifuge tubes for ultracentrifugation.

  4. Temperature-controlled tabletop centrifuge

  5. Electrophoresis system suitable for agarose gel

  6. A spectrometer capable of measuring UV absorbance, as well as matching quartz cuvettes

  7. A short wavelength UV light

  8. Pipette as well as matching pipetting tips. Any kind of pipette (manual, automatic, serological, etc.) works as long as they meet the requirement and are fit for use

  9. Liquid nitrogen tank (and liquid nitrogen supply) or -80 °C freezer for storing bacterial strains

  10. Incubator for bacterial growth

Procedure

  1. Preparation of -80 °C cell pellets

    1. Grow R. palustris cultures as required by the experimental design. Typical growth conditions that we use are given in Kim and Harwood (1991). However, the composition of the cultivation medium is not important for the protocol we describe here. It also does not matter if cells are grown aerobically or anaerobically. For one typical sample, we usually use 50-70 ml of culture at Abs660 ~1 (medium is used as blank).

    2. Add 10 ml of RNase-free water to one 50-ml centrifuge tube. Place the tube horizontally in a freezer (any sub-zero °C freezer would work) to form a thin sheet of ice (we usually do this a few days in advance to make sure ice is formed). One tube with ice sheet would be used collect ~35 ml of cell culture. Prepare as many tubes as required in advance.

    3. When the culture reaches the desired growth state, pour the culture directly into the tube containing the ice sheet and place the tube on ice. The purpose of this step is to minimize the time required for the culture to cool down and thus capture the native form of ribosomes. It is also referred as “fast-cool” in some literature ( Lawrence et al., 2016 ).

    4. Invert the tubes with culture gently from time to time to facilitate the cooling down of the culture. We usually transfer the now cold culture into a pre-chilled centrifuge tube. This process usually takes 5-10 min in our experience.

    5. Spin at ~5,000 x g for 10 min at 4 °C to collect the cell pellets.

    6. Discard the supernatant and freeze the pellets with liquid nitrogen. The frozen cell pellets can now be stored at -80 °C. In our experience, the ribosome profile of the sample can be preserved for 6 weeks at least.

    Note: For each sample, we usually collect cells from ~70 ml culture and store the pellets in two 50-ml centrifuge tubes. This can be easily scaled up or down depending on the need of sampling and the availability of equipment.


  2. Purification of ribosomes

    1. Prepare two screw-capped 2 ml centrifuge tubes for each sample (assuming the pellets are collected in two 50 ml centrifuge tubes, see the Note in Procedure A). Add ~500 µl Zirconia/Silica beads to each tube and autoclave all of the tubes.

    2. Resuspend the cell pellet in 1.2 ml cold lysis buffer in each 50 ml centrifuge tube. Use pipet to mix well and transfer the resuspended sample to the 2 ml screw-cap tube containing Zirconia/Silica beads.

    3. In a 4 °C room, lyse the resuspended cells with a Biospec Mini-Beadbeater-24 at 3,500 rpm for 1 min and then chill them on ice for 1min. Repeat 3 more times, 4 times in total.

      Note: The setup of beadbeater cycle, speed and time may need to be adjusted according to the species of interest.

    4. Centrifuge for 10 min at 20,000 x g with a tabletop microcentrifuge at 4 °C to remove the beads and cell debris.

    5. Collect 1 ml of supernatant from each tube. Combine supernatants from the same sample if needed (2 ml of supernatant for each sample in our setting, see the Note in Procedure A). Centrifuge for 30 min at 30,000 x g at 4 °C with an ultracentrifuge to further clarify the lysates. Collect the supernatant.

    6. Centrifuge the supernatant from last step at 100,000 x g with an ultracentrifuge for four hours at 4 °C to pellet the ribosomes.

    7. After the spin, carefully remove the supernatant by pipetting without disturbing the pelleted ribosomes. The pelleted ribosomes, when abundant, visually resemble a contact lens.

    8. Without disturbing the pellets, carefully add 200-500 µl Buffer A1 to the centrifuge tube containing the pelleted ribosomes. Leave the samples on ice overnight to allow the ribosomes to dissolve in the buffer.

    9. On the next day, gently mix the dissolved ribosomes. Take a sample and determine the absorbance at 260 nm (A260). The volume of the sample taken depends on the concentration and the setup of spectrometer. The sample can also be diluted if necessary. In our experience, a typical yield from one sample is 400 µl of 20 relative units (R.U.) of A260 in total. Note that the yield can vary significantly depending on the experiment.

    10. Adjust all the samples to the same concentration. The minimum concentration of ribosome for the following analysis is 50 R.U. of A260/ml. Freeze the purified ribosomes with liquid nitrogen and store at -80 °C for further analysis. The purified ribosomes are suitable for further assays such as separation on a sucrose gradient or for isolation and analysis of rRNA associated with ribosomes.


  3. Analyze rRNA by electrophoresis and Northern blot.

    This procedure can be applied to total RNA isolated from cultures or from purified ribosomes. RNA is purified following either a QIAzol-based method (see Yin and Harwood, 2020) or the commercially available protocol for the miRNeasy Mini Kit (Qiagen). For the QIAzol-based method, we typically start with 400 µl of an 0.1 absorbance (RU of A260) preparation of ribosomes and the method yields a 15 µl volume of approximately 500 ng RNA/µl.

        An optional step is to use an RNA ScreenTape System (Agilent) to get a snapshot of the rRNA profile. To specifically detect rRNA by Northern blot, rRNA has to be first separated by electrophoresis on an agarose gel. Since agarose gels are usually too thick to be used in the commonly used electro-transfer system, this calls for a capillary transfer system (upward or downward) to transfer RNA from the gel to a membrane for subsequent Northern blot detection. This procedure will be described here in detail. For Northern blot detection, we use a non-radioactive system and follow its commercially available manual (DIG). This greatly saves time and effort as described in detail in the accompanying protocol “Charging state analysis of transfer RNA from an α-proteobacterium” (Yin and Harwood, 2020). The principle of designing and generating a DIG-labeled probe is also described there.


    1. In a 50-ml centrifuge tube, add 0.2 g agarose to 20 ml 1x TBE buffer. Melt agarose in a microwave and pour the gel.

    2. Mix 1 µg total RNA with 2x loading dye. Heat at 70 °C for 10 min. Immediately chill on ice for 3 min. Spin briefly.

    3. Analyze the sample on an agarose gel with a running buffer of 1x TBE buffer. For a gel of ~10 cm in length, a one-hour run at 100 V usually achieves satisfactory separation. After electrophoresis with a setting like this,16S rRNA is usually in the middle of the gel.

    4. After the electrophoresis, immerse the gel in 20x SSC twice, each time for 10 min.

    5. Assemble the transfer apparatus as follows:

      Top

      Light weight such as a pipet tip box

      1 Whatman filter paper connecting to the reservoir, immersed in 20x SSC

      1 Whatman filter paper, slightly bigger than the gel, immersed in 20x SSC

      Agarose gel, the side with RNA facing down

      Membrane cut to gel size, pre-immersed in 20x SSC

      2 Whatman filter papers, slightly bigger than the gel, immersed in 20x SSC

      Dry paper towels (~20 sheets at least)

      Bottom

      Note: This describes the transfer apparatus for downward transfer. Slightly modified setup can be used for upward transfer.

      Set up the capillary transfer system as shown in Figure 1. Leave on bench overnight.



      Figure 1. Capillary transfer system (RNA transferred downward from gel to membrane)


    6. The membrane can be cross-linked the next morning. One option to cross-link is to place the membrane 1-2 cm from a shortwave UV light, the side with sample facing UV light, and expose it to UV for 10 min. Next, follow commercially available protocol for DIG system for Northern blot (Sessions in Hybridization and Immunological detection in https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/12039672910bul.pdf).

Data analysis

An example of 16S rRNA Northern blot is shown in Figure 2. Total RNA isolated from wild-type (WT) and a mutated (mutant) R. palustris are analyzed by RNAtape and Northern blot. The RNAtape result shows an extra band immediately above the 16S rRNA from the mutant. This band was subsequently identified as an intermediate product of 16S rRNA synthesis, as it is identified in Northern blot for 16S rRNA. Detailed analysis of rRNA Northern blots can be found in Figures 6 and 7 fromYin et al. (2019).


Figure 2. An example of 16S rRNA Northern blot. RNA isolated from wild type (WT) on RNA tape shows typical 23S rRNA, 16S rRNA and other RNA species. RNA isolated from mutant with defective ribosome biosynthesis on RNA tape shows an extra band immediately above 16S rRNA (asterisk) on RNA tape. Northern blot using a probe targeting 16S rRNA detects the 16S rRNA in WT and the mutant, as well as the extra band immediately above 16S rRNA. The result is consistent with the hypothesis that the extra band is an intermediate product of 16S rRNA biosynthesis. Note that in the figure above, our purpose was to determine the nature of irregular band labeled with asterisk, hence more total RNA from the mutant was analyzed in Northern blot comparing to the WT. Same amount of total RNA was analyzed on RNA tape in the figure above.

Recipes

Note: The reagents listed here below are RNase free. The working bench should be cleaned with RNaseZap or equivalent to decontaminate RNase before use.


  1. Buffer A1 (10x)

    200 mM HEPES-KOH, pH 7.5

    300 mM NaCl

    80 mM MgCl2

    Notes:

    1. Buffer A1 is one of the most critical items in ribosome purification. When analyzed in vitro, ribosome profiles are very sensitive to the buffer components. For example, if MgCl2 is reduced to 60 mM in 10x Buffer A1, the population of 70S ribosome will be greatly reduced in sucrose gradient analysis. Higher MgCl2 concentrations are also sometimes used to better preserve the population of 100S ribosome. Therefore, one should be cautious about interpreting results from assays such as sucrose gradients, especially with organisms that are less commonly studied. A useful control is to purify ribosomes from E. coli with the buffers to be used, and then examine whether they behave as expected.

    2. On the same note, additional consideration should be given to the enzyme activities needed to be preserved if purified ribosomes are to be used in assays such as in vitro translation. The recipe here is sufficient to preserve the profile of ribosomes during the purification process, namely the population of 30S, 50S, 70S and 100S ribosomes. We encourage the reader to do a preliminary study as well as a broader literature review to determine the optimal buffer components for their organism, system and question of interest (Lawrence et al., 2016; Basu and Yap, 2017).

  2. Lysis buffer

    Add RNasin into Buffer A1 (1x) to a final concentration of 1 µl/ml

Acknowledgments

This work was supported by grant W911NF-15-1-0150 to C.S.H. from the U.S. Army Research Office. We thank Prof. David Morris, University of Washington, for his help with ribosome profiling. This protocol is derived from our previous work published in 2019 (Yin et al., 2019).

Competing interests

The authors claim no financial or non-financial competing interests.

References

  1. Basu, A. and Yap, M. N. (2017). Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase. Proc Natl Acad Sci U S A 114(39): E8165-E8173.
  2. Kim, M. K. and Harwood, C. S. (1991). Regulation of benzoate-CoA ligase in Rhodopseudomonas palustris. FEMS Microbiology Letters 83: 199-203.
  3. Lawrence, M. G., Shamsuzzaman, M., Kondopaka, M., Pascual, C., Zengel, J. M. and Lindahl, L. (2016). The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation. Nucleic Acids Res 44(12): 5798-5810.
  4. Yin, L., Ma, H., Nakayasu, E. S., Payne, S. H., Morris, D. R. and Harwood, C. S. (2019). Bacterial longevity requires protein synthesis and a stringent response. mBio 10(5).
  5. Yin, L. and Harwood, C. S. (2020). Charging State Analysis of Transfer RNA from an α-proteobacterium. Bio-protocol 10(23): e3834.

简介

[摘要]核糖体是细胞生命的组成部分。它们是由多种核糖体蛋白和RNA组成的复杂分子机器。为了研究核糖体组成的不同方面,几十年来已经开发了许多方法。在这里,我们描述如何从α-变形杆菌Rhodopseudomonas palustris中纯化核糖体。按照该协议,可以从纯化的核糖体中提取RNA,也可以直接从细胞培养物中提取RNA,并使用Northern印迹对核糖体RNA进行定量。该协议给出了研究除常用大肠杆菌外的细菌中核糖体的一个例子。还详细介绍了使用rRNA进行Northern杂交的挑战。

[背景]细菌细胞的命运是紧密相连的核糖体。我们最近的研究表明,活性核糖体在营养缺乏的palustris细胞的存活机制中起着重要作用(Yin等人,2019)。核糖体通过一系列超速离心纯化,并采用经典方法优化的方法(Lawrence等,2016)。使用不太常用的毛细管转移系统,通过RNA印迹检测核糖体RNA群体。纯化步骤的细节可能极大地影响核糖体的状态。这些方法在这里进行了详细描述,对于研究多种细菌中的翻译设备的研究人员应该具有广泛的兴趣。

关键字:Ribosome profile, 毛细管转移, 核糖体RNA, Northern印迹, 沼泽红假单胞菌

材料和试剂

注意:所有试剂都可以从头生产或从不同的供应商处购买,只要它们不含RNase。
1.不含RNase的离心管(ThermoFisher AM12400或等效产品)。使用与超速离心机兼容的试管进行超速离心(更多信息,请参阅读者的超速离心机的手册)。     
2. 2毫升螺口盖管(Sigma - Aldrich,BR780758或同等水平)     
3. Costar无菌一次性试剂容器(Fisher Scientific,目录号:07-200-128)     
4. HEPES-KOH,1 M,pH 7.5(波士顿生物产品,目录号:BBH-75-K)     
5. MgCl 2,1 M(波士顿生物产品,目录号:BM-670)     
6. NaCl,分子生物学等级(Promega Corp,目录号:H5271)     
7.重组RNasin核糖核酸酶抑制剂(Promega Corp,目录号:N2511)     
8. miRNeasy迷你套件(Qiagen,目录号:217004)     
9.琼脂糖,分子生物学等级     
10. UltraPure不含DNase / RNase的蒸馏水(Invitrogen,目录号:10977-023) 
11. Amersha m Hybond -XL膜(GE生命科学) 
12. 10个Tris-borate- EDTA(TBE)缓冲液(Thermo Fisher Scientific,目录号:AM9863) 
13. 20x SSC缓冲区(Sigma - Aldrich,目录号:S6639) 
14. DIG RNA标记混合物(Sigma - Aldrich,目录号:11277073910) 
15. DIG Easy Hyb (Sigma - Aldrich,目录号:11603558001) 
16. DIG洗涤和封闭缓冲液组(Sigma - Aldrich,目录号:11585762001) 
17.抗地高辛配基-AP,Fab片段(Sigma-Aldrich,目录号:11093274910) 
18. CDP星(Sigma-Aldrich,目录号:11685627001) 
19. RNA凝胶上样染料(2×)(赛默飞,Ç atalog号:R0641) 
20.缓冲区A1(10x)(请参阅配方) 
21.裂解缓冲液(请参见食谱) 

设备

注意:在使用前,应使用RNaseZap或等同物对RNase进行净化,以清洗工作台以及电泳,转移和Northern blot系统。可以从任何可靠的供应商处获得下面未列出目录号的物品。
微型珠磨器-24(生物规格PRODUCTS INC 。,目录号:112011)
氧化锆/二氧化硅珠,0.1毫米直径(生物规格PRODUCTS INC 。,目录号:11079101z )
Beckman TL-1000超速离心机(已停产的型号,可以用具有温度控制功能的等效超速离心机代替)。请确保使用兼容的离心管进行超速离心。
温控台式离心机
适用于琼脂糖凝胶的电泳系统
能够测量紫外线吸收率的光谱仪以及配套的石英比色皿
短波长紫外线
移液器以及匹配的移液器吸头。只要满足要求并适合使用,任何类型的移液器(手动,自动,血清学等)都可以工作
液氮罐(和液氮供应)或-80 °C的冰箱用于存储细菌菌株
细菌生长培养箱

程序

-80 °C细胞沉淀的制备
增长沼泽红假单胞菌立方米ltures一个Ş要求的实验设计。我们使用的典型生长条件在Kim和Harwood (1991)中给出。但是,培养基的组成对于我们在此描述的方案并不重要。如果细胞需氧或厌氧生长,它也不会发生。对于一个典型的样品,我们通常在50至70 ml Abs 660〜1的培养液中使用(培养基用作空白)。
将10 ml无RNase的水加到一个50 ml的离心管中。将试管水平放置在冰箱中(任何低于0 °C的冰箱都可以使用)以形成薄薄的冰片(我们通常提前几天进行操作以确保形成冰)。ö用冰片NE管将用于收集〜35毫升细胞培养物。预先准备所需数量的试管。
当培养物达到所需的生长状态时,将培养物直接倒入装有冰盖的试管中,然后将其放在冰上。该步骤的目的是使培养物冷却并因此捕获天然形式的核糖体所需的时间最小化。在某些文献中,它也被称为“快冷” (Lawrence等,2016)。
时不时轻轻地将培养管倒置,以促进培养液的冷却。我们通常将现在冷的培养物转移到预冷的离心管中。根据我们的经验,此过程通常需要5-10分钟。
在4 °C下以〜5,000 x g旋转10分钟以收集细胞沉淀。
丢弃上清液,并用液氮冷冻沉淀。现在可以将冷冻的细胞沉淀物储存在-80 °C下。根据我们的经验,样品的核糖体特征至少可以保留6周。
注意:对于每个样品,我们通常从约70 ml培养物中收集细胞,并将沉淀物存储在两个50 ml离心管中。根据采样的需要和设备的可用性,可以轻松地按比例放大或缩小比例。

核糖体的纯化
为每个样品准备两个螺口盖的2 ml离心管(假设将沉淀物收集在两个50 ml离心管中,请参见步骤A中的注释)。在每个试管中加入〜500 µl氧化锆/二氧化硅珠,并对所有试管进行高压灭菌。
在每个50 ml离心管中的1.2 ml冷裂解缓冲液中重悬细胞沉淀。用移液器充分混合,然后将重悬的样品转移至装有氧化锆/二氧化硅珠的2 ml螺帽管中。
在4 ℃下室,细胞溶解的重悬细胞与Biospec微型珠磨器-24以3,500rpm进行1分钟,然后放松它们在冰上1分钟。再重复3次,总共重复4次。
注:牛逼,他建立珠磨周期,速度和时间可能需要根据感兴趣的种类进行调整。
用台式微量离心机在4 °C下以20,000 xg离心10分钟,以除去珠子和细胞碎片。
从每个管中收集1 ml上清液。如果需要,合并来自同一样品的上清液(在我们的设置中,每个样品2 ml上清液,请参见步骤A中的注释)。在4 °C下以3 0,000 xg离心30分钟,以进一步澄清裂解物。收集上清液。
最后一步将上清液以100,000 xg离心,并在4 °C下超速离心4小时以沉淀核糖体。
旋转后,通过移液小心除去上清,而不会干扰颗粒状核糖体。当颗粒状核糖体丰富时,在视觉上类似于隐形眼镜。
在不干扰沉淀的情况下,小心地将200-500 µl Buffer A1添加到装有沉淀的核糖体的离心管中。将样品放在冰上过夜,以使核糖体溶解在缓冲液中。
第二天,轻轻混合溶解的核糖体。取样并测定260 nm(A 260 )的吸光度。所取样品的体积取决于浓度和光谱仪的设置。如果需要,样品也可以稀释。根据我们的经验,一个样品的典型产量为400微升,共20个相对单位(RU)A 260 。请注意,产量可能会因实验而有很大差异。
将所有样品调整至相同浓度。用于以下分析的最小核糖体浓度为50 RU A 260 / ml 。用液氮冷冻纯化的核糖体,并储存在-80 °C以便进一步分析。纯化的核糖体适用于进一步的测定,例如在蔗糖梯度上分离或用于分离和分析与核糖体相关的rRNA。

通过电泳和RNA印迹分析rRNA。
该程序可应用于从培养物或纯化的核糖体中分离的总RNA。RNA被eithe以下纯化为r的Qiazol加基础的方法(参见阴和哈伍德,2020 )或对市售协议miRNeasy Mini试剂盒(Qiagen)进行。对于Qiazol加基础的方法中,我们通常先从400 μ的0.1吸光度升(A260的RU)制备核糖体的,并且该方法产率小号1 5微升的体积大约500ng的RNA /微升。
  可选步骤是使用RNA ScreenTape系统(安捷伦)获取rRNA配置文件的快照。为了通过Northern印迹法特异性检测rR NA,必须先在琼脂糖凝胶上通过电泳分离rRNA。由于琼脂糖凝胶通常太粘稠,无法在常用的电转移系统中使用,因此需要毛细管转移系统(向上或向下)将RNA从凝胶转移到膜上,以便随后进行Northern印迹检测。此处将详细描述此过程。对于Northern印迹检测,我们使用非放射性系统并遵循其市售手册(DIG)。如所附协议“从α-变形杆菌中转移RNA的电荷状态分析”中详细描述的那样,这极大地节省了时间和精力。那里还描述了设计和生成DIG标记探针的原理。

在50毫升离心管中,将0.2克琼脂糖加入20毫升1x TBE缓冲液中。在微波炉中融化琼脂糖,倒入凝胶。
将1 µg总RNA与2x上样染料混合。在70 °C下加热10分钟。立即在冰上冷却3分钟。短暂旋转。
使用1x TBE缓冲液运行缓冲液在琼脂糖凝胶上分析样品。对于约10厘米长的凝胶,在100 V下运行1小时通常可以达到令人满意的分离效果。经过这样的设置后,16S rRNA通常位于凝胶的中间。
电泳后,将凝胶浸入20x SSC两次,每次10分钟。
组装传输设备如下:
              最佳
重量轻,例如移液器吸头盒
1张Whatman滤纸连接到水箱,浸入20倍SSC中
1张Whatman滤纸,比凝胶稍大,浸入20倍SSC
琼脂糖凝胶,RNA朝下的一面
将膜切成凝胶大小,预先浸入20x SSC中
2张Whatman滤纸,比凝胶稍大,浸入20倍SSC
干纸巾(至少约20张)
底部
注意:这描述了向下传送的传送设备。稍微修改的设置可以用于向上传输。

如图1所示设置毛细管传输系统。



图1.毛细管转移系统(RNA从凝胶向下转移至膜)

该膜可以在第二天早晨交联。交联的一种选择是将膜放置在离短波紫外线1至2厘米处,使样品面向紫外线的一面,并将其暴露于紫外线10分钟。接下来,按照用于DIG系统进行Northern杂交的商业协议进行操作(在https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/12039672910bul.pdf中进行杂交和免疫学检测) 。


数据分析

的的16S rRNA Northern印迹的例子在图2中示出的总RNA从野生型(WT)和突变(突变体)分离沼泽红假单胞菌通过分析RNAtape和Northern印迹。所述RNAtape结果表明额外的带立即从突变体中的16S rRNA的上方。该条带随后被鉴定为16S rRNA合成的中间产物,因为它在Northern blot中被鉴定为16S rRNA。可从Yin等人的图6和7中找到rRNA Northern印迹的详细分析。(2019)。



图2. 16S rRNA Northern印迹的一个例子。从RNA胶带上的野生型(WT)分离的RNA显示典型的23 S rRNA,16 S rRNA和其他RNA种类。从RNA磁带上具有不良核糖体生物合成缺陷的突变体中分离出的RNA在RNA磁带上的16 S rRNA(星号)上方显示了一个额外的条带。使用靶向16 S rRNA的探针进行Northern blot检测WT和突变体中的16 S rRNA,以及16 S rRNA上方的额外条带。该结果与以下假设相符:多余的条带是16 S rRNA生物合成的中间产物。请注意,在上图中,我们的目的是确定标有星号的不规则条带的性质,因此与WT相比,在Northern印迹中分析了来自该突变体的更多总RNA。上图中,在RNA胶带上分析了相同量的总RNA 。

菜谱

注:在这里列出的试剂下面是不含核糖核酸酶。在使用前,应使用RNaseZap或类似的工具清洁工作台以净化RNase。

缓冲区A1 (10x)
200 mM HEPES-KOH,pH 7.5
300毫米氯化钠
80毫米MgCl 2
注意小号:
缓冲液A1是核糖体纯化中最关键的项目之一。在体外分析时,核糖体谱对缓冲液成分非常敏感。例如,如果在10x Buffer A1中将MgCl 2还原为60 mM,那么在蔗糖梯度分析中70S核糖体的数量将大大减少。有时还使用较高的MgCl 2浓度来更好地保护100S核糖体。因此,在解释诸如蔗糖梯度之类的测定法的结果时应特别谨慎,尤其是对于那些研究较少的生物而言。一个有用的控制措施是使用要使用的缓冲液从大肠杆菌中纯化核糖体,然后检查它们的行为是否符合预期。
同样,如果将纯化的核糖体用于测定(如体外翻译)中,则应进一步考虑需要保留的酶活性。这里的配方足以在纯化过程中保留核糖体的特征,即30S,50S,70S和100S核糖体的数量。我们鼓励读者进行初步研究以及更广泛的文献综述,以确定针对其生物体,系统和感兴趣的问题的最佳缓冲液成分(Lawrence等,2016; Basu和Yap,2017)。
裂解缓冲液
将RNasin加入缓冲液A1(1x)中,终浓度为1 µl / ml

致谢

这项工作得到了美国陆军研究办公室授予CSH的W911NF-15-1-0150资助。我们感谢华盛顿大学的David Morris教授在核糖体分析方面的帮助。该协议源自我们先前于2019年发表的工作(Yin等人,2019)。

利益争夺

作者声称没有任何金融或非金融竞争利益。

参考文献

Basu ,A.和Yap,MN(2017)。进化上保守的GTPase的冬眠100S核糖体的金黄色葡萄球菌的拆卸。PROC国家科科学院科学USA 114(39):E8165-E8173。
金,M 。ķ 。和Harwood,C.S 。(1991)。暗纹假单胞菌中苯甲酸酯-辅酶A连接酶的调控。FEMS微生物学快报83:199-203。
MG的劳伦斯(Lawrence,MG),M。的Shamsuzzaman ,M。的Kondopaka ,C。的Pascual,Zengel ,JM和L.Lindahl (2016)。大肠杆菌核糖体蛋白uL4和uL22的延伸环有助于核糖体组装和蛋白质翻译。Nucleic Acids Res 44(12):5798-5810。
Yin,L.,Ma,H.,Nakayasu ,ES,Payne,SH,Morris,DR和Harwood,CS(2019)。细菌寿命需要蛋白质合成和严格的反应。mBio 10(5)。              
Yin,L.和Harwood,CS(2020)。来自α-变形杆菌的转移RNA的充电状态分析。生物协议10(23):e3834。
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引用:Yin, L. and Harwood, C. S. (2020). Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot. Bio-protocol 10(23): e3835. DOI: 10.21769/BioProtoc.3835.
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