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Oct 2019

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Charging State Analysis of Transfer RNA from an α-proteobacterium
α-变形杆菌转运RNA的装载状态分析   

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Abstract

Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces the time and the effort required for this protocol.

Keywords: Translation (翻译), tRNA charging (tRNA装载), Aminoacyl-tRNA (氨酰转移RNA), Acid urea gel (醋酸尿素凝胶), Rhodopseudomonas palustris (沼泽红假单胞菌)

Background

The primary function of tRNA is, with the help of other translation factors, to ensure the accurate translation of mRNA to protein. Aminoacyl-tRNAs (charged) bring amino acids to the ribosome for peptide elongation, and then the uncharged tRNAs are released. The charging state of tRNA is largely determined by the available resource (i.e., amino acids) and their consumption by the ribosome. To analyze the charging state of cellular tRNA, methods have been developed using acid-urea gels to separate the total RNA and to detect the tRNA of interest by Northern blot (Janssen et al., 2012; Bullwinkle and Ibba, 2016). Recently, we explored the relationship between tRNA charging states and survival of starving bacteria (Yin et al., 2019). The method used in that study and elaborated here, has optimized the RNA purification step to better capture the tRNA charging state in bacteria other than E. coli. It also utilizes a commercially available system to perform the analysis without using radioactive materials, which could be helpful for some research groups.

Materials and Reagents

Notes:

  1. It is crucial to keep all the materials and reagents RNase-free for this protocol. To this end, the reagents and materials are all RNase-free, and are purchased pre-made or ready-to-dissolve in order to simplify the procedure as much as possible. Disposable RNase-free centrifuge tubes are used to make reagents whenever applicable.

  2. Multiple kits are available for DIG reagents. Items 19-23 are listed here for readers to choose from to suit the scale required.


  1. Centrifuge tubes, RNase free (1.5 ml, 2 ml, 15 ml and 50 ml, as needed)

  2. QIAzol Lysis Reagent (Qiagen, catalog number: 79306)

  3. Chloroform, molecular biology grade

  4. Isopropanol, molecular biology grade

  5. Ethanol, molecular biology grade

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

  7. TEMED (National Diagnostics, catalog number: EC-503)

  8. Ammonium persulfate (National Diagnostics, catalog number: EC-504)

  9. Urea (National Diagnostics, catalog number: EC-605)

  10. AccuGel 29:1 (30%) (National Diagnostics, catalog number: EC-851)

  11. Sodium acetate, 3 M solution, pH 4.5 (National Diagnostics, catalog number: EC-905)

  12. Sodium acetate, 3 M solution, pH 5.2 (National Diagnostics, catalog number: EC-906)

  13. EDTA, 0.5 M, pH 8.0 (Thermo Fisher Scientific, catalog number: AM9260G)

  14. Xylene cyanol, molecular biology grade

  15. Bromophenol blue, molecular biology grade

  16. Tris-HCl solution, 1 M, pH 9.0 (Sigma-Aldrich, catalog number: T2819-100ML)

  17. Amersham Hybond-XL membrane (GE Life Sciences, catalog number: 45-001-148)

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

  19. DIG RNA labeling Mix (Sigma-Aldrich, catalog number: 11277073910)

  20. DIG RNA labeling kit (Roche, catalog number: 11175025910)

  21. DIG system (Roche, catalog number: 12039672910)

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

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

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

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

  26. RNase Zap (Thermo Fisher, catalog number: AM9780)

  27. Growth medium for R. palustris (PM medium) (see Recipes)

  28. Acid-urea gel (see Recipes)

  29. Sodium acetate-EDTA running buffer (see Recipes)

  30. Sodium acetate-EDTA loading 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.


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

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

  3. Electrophoresis, transfer and Northern blot system that fits a gel 20 cm in height

  4. Temperature-controlled tabletop centrifuge

  5. PCR machine for temperature-controlled incubation or equivalent incubator

Software

  1. ImageJ

Procedure

  1. Isolation of total RNA from R. palustris

    Notes:

    1. It is crucial to choose the proper cell lysis method for the organism of interest in order to capture the native charging state of tRNA. The method described here, based on micro beads and a beadbeater, is routinely used in our group to isolate RNA from R. palustris and related α-proteobacteria. We strongly advise the reader to test different lysis methods for their organism of interest (e.g., the ratio between bead volume and medium volume, the number of cell beating cycles, etc.).

    2. Another note about tRNA charging states is that, as elaborated elsewhere (Janssen et al., 2012; Bullwinkle and Ibba, 2016), the sample needs to be kept in acidic conditions all the time. Acidic buffers and gel systems are used throughout this protocol.

    3. RNA purified from Procedure A can be used directly in the following steps in this protocol. This is because the charging state of tRNA is calculated based on the ratio of charged vs uncharged tRNA. Slight contamination of DNA and/or protein, therefore, will not affect the end result after the purification described here. If the purified RNA is to be used in other procedure such as RNAseq or in vitro translation, further purification would be required.


    1. Before the experiment, prepare one screw-capped 2 ml centrifuge tube for each sample. Add ~500 µl of Zirconia/Silica Beads into each tube and autoclave all of the tubes.

    2. Grow R. palustris cultures as required by the experimental question. 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. The typical sample contains 3-5 ml of culture at Abs660 ~1.2. Collect the cells at 4 °C by centrifugation at 4,000 rpm and store the pellets at -80 °C.

    3. Resuspend each frozen cell pellet in 1 ml of QIAzol Lysis Reagent. Mix gently with pipetting and transfer the resuspended samples into the tubes with beads. This step should be performed on ice.

    4. Break the cells with a beadbeater at 3,500 rpm for 1 min and then place the samples on ice for another 1 min. Repeat this cycle 3 more times.

    5. Place all the samples at room temperature for 5 min.

    6. Add 200 µl chloroform to each sample. Shake the tube vigorously by hand for 15 s.

    7. Place the tube at room temperature for 3 min.

    8. Centrifuge the samples for 15 min at 12,000 x g at 4 °C. Carefully transfer 560 µl of the colorless upper aqueous phase to a new 1.5 ml centrifuge tube.

    9. Add 0.5 ml isopropanol and mix thoroughly by vortexing.

    10. Centrifuge for 15 min at 12,000 x g at 4 °C. Discard the supernatant.

    11. Add 1 ml of 75% ethanol. Centrifuge for 5 min at 7,500 x g at 4 °C.

    12. Remove the supernatant completely. Air-dry the RNA pellet in a fume hood briefly (5-10 min).

    13. Re-dissolve each RNA pellet in 10 mM of sodium acetate, pH 4.5. The purified RNA can be stored at -80 °C for at least two weeks. A typical yield is 50 µl of 5 µg/µl total RNA. This number can vary depending on factors such as species, growth states and sampling volume.


  2. Alkaline pH treatment to produce a deacylated (uncharged) tRNA control

    1. Take ~10 µg of purified RNA. Add stock solution of 0.5 M EDTA and 1 M Tris-HCl (pH 9.0) directly to the sample in order to adjust the final buffer to 1 mM EDTA and 100 mM Tris-HCl, pH 9.0. Adjust final volume to 50 µl. Mix well by pipetting.

    2. Incubate the sample at 42 °C for 60 min.

    3. Add 5 µl of 3 M sodium acetate, pH 5.2 to the sample. Reverting the buffer back to pH 5.2 will not make the sample acylated again.

    4. Add 137.5 µl of ice-cold 95% ethanol to the sample, then incubate on ice for 10 min.

    5. Centrifuge at 13,000 x g for 10 min at 4 °C to precipitate RNA.

    6. Remove the supernatant and wash the RNA pellet with 1 ml of 70% ethanol.

    7. Centrifuge at 13,000 x g for 10 min at 4 °C.

    8. Remove the supernatant completely. Air-dry the RNA pellet as needed.

    9. Dissolve deacylated RNA with ~30 µl 10 mM sodium acetate, pH 4.5.


  3. Making DIG-labeled probe for tRNA-Trp

    DIG RNA labeling kit was used in our project to generate labeled probe for subsequent experiments. Equivalent reagents, such as DNase I and T7 RNA polymerase, can be substituted where suitable. Other strategies such as the classical P32-labeling can also be used for this step. If so, the following experiments should be adjusted accordingly. As the detailed protocol for the DIG kit is commercially available, this section will focus on how we adapted it to detect tRNATrp in R. palustris CGA009.


    1. A stretch of 31 nucleotides in R. palustris CGA009 tRNATrp was chosen as the target of the probe. The following dsDNA was commercially made (IDT) as the template of in vitro transcription:

      TGAATTGTAATACGACTCACTATAGGGGGTTTTGGAGACCGGTGCTCTACCAATTGAG

      A basic purification level, which should be molecular biology grade by default, was sufficient for dsDNA in our experience. We have also ordered two complementing ssDNA and annealed them together to form the dsDNA (see Reference 5 for more information), and it worked as well.

          Within the above sequence, the shaded part is the promoter for T7 RNA polymerase and the underscored part is the reverse-complement of our target on tRNATrp. A 7-bp clamp is added before the promoter to facilitate polymerase binding. The selection of tRNA target-sequence needs to be done case by case and the readers are encouraged to try several candidate sequences.

    2. Make the reaction mixture following the instructions for the DIG RNA labeling kit. Below is an example we used for our project (all reagents here except the template are included in the kit). The volume can be scaled up or down as needed.

      9 µl RNase-free water

      4 µl transcription (5x) buffer

      4 µl DIG labeling mix

      2 µl T7 polymerase

      1 µl template of dsDNA (10 ng/µl is sufficient)

    3. Incubate the reaction mixture at 42 °C for 1.5 h.

    4. For each reaction, add 2 µl DNaseI and incubate at 37 °C for 15 min.

    There is no need to further purify the probe. The reaction product is diluted and ready for further steps. A testing Northern blot, such as a dot blot with total RNA, can be used to determine the optimal dilution factor for the probe to be used in actual experiments (e.g., make a serial dilution of the probe by the factor of 10, then test which concentration generates the best signal-noise ratio in reader’s detection system).


  4. Acid-urea polyacrylamide gel electrophoresis

    An electrophoresis system of at least 20 cm in height is recommended to analyze the charging states of tRNAs. The thickness of gel/spacer does not matter as long as the well can be sufficiently cleaned of urea. The difference between charged and uncharged tRNA is small in size. A smaller gel might be suitable to analyze some peptidyl-tRNAs, but in general does not provide the resolution to analyze aminoacyl-tRNAs. For each run, a sample of uncharged tRNA must be included as the negative control.

    1. Assemble the electrophoresis system with acid-urea gel. Add the cold sodium acetate-EDTA running buffer and pre-run the gel at 20 V for 30 min at 4 °C. The purpose of pre-running is to bring up the temperature of the gel. See more information at company’s page (see Reference 6).

    2. Incubate the sample at 72 °C for 3 min. Briefly spin the sample and mix it with the loading buffer at a 1:1 ratio. We usually loaded ~20 µl of sample, a total of 2.5-5 µg total tRNA, per well for a gel of 20 cm height. The minimum and maximum amounts of sample required need to be tested and determined case by case.

    3. Clean the loading wells with syringe and needle. This needs to be done immediately before applying the sample to the well. For acid-urea gel, urea continues to accumulate in the well. This can prevent the sample from settling at the bottom of the well if not properly cleaned.

    4. Load the sample. There should be no change of dye color. Run the gel at 15 W for 12 h (for a gel of 20 cm height) at 4 °C. It is crucial to bring up and maintain the operating temperature of the gel during the run, hence the setting of constant power as well as the pre-run specified above (see Reference 5 for more information).


  5. Northern blot analysis

    1. After the electrophoresis, any standard protocol suiting the available system can be used to transfer RNA from the gel to a membrane. In our case, we used 0.5x TBE as the transfer buffer. We did a standard wet-transfer was at 4 °C, either at 20 V overnight or at 650 mA for 30 min. No obvious difference was observed between the two settings. The membrane is then ready for cross-link and following Northern blot. In our experience, exposure to a UV-light box for 10-15 min was sufficient and could serve as a start point.

    2. The standard protocol of the DIG system (Roche, see Reference 7 for more information) was used for Northern blot blocking, washing and detection steps. Briefly, the probe for tRNATrp is used to detect tRNATrp on the membrane, followed by interaction with Anti-Digoxigenin-AP and then CDP-Star to generate chemiluminescence signals. Any suitable imaging system, such as film or digital imager, can be used to detect and quantify the results.

Data analysis

ImageJ was used for quantification of the image on films. For results obtained with a digital imager, the accompanying software was used for quantification. The charging rate of tRNA is calculated as:

A brief walkthrough of tRNA charging gel analysis is provided here. As shown in Figure 1; lane 1 contains untreated total RNA from growing R. palustris, which mainly contains charged tRNA. The same sample is deacylated as described in this protocol and analyzed in lane 2. The sample to be analyzed is in lane 3. The bands corresponding to charged and uncharged tRNA are determined by referring to the bands in lanes 1 and 2. The intensity of the individual bands is the analyzed with ImageJ. An example can also be found in Figure 5d and 5e from Yin et al. (2019).



Figure 1. Northern blot showing separation of charged and unchaged tRNATrp

Recipes

  1. Growth medium for R. palustris (PM medium)

    For 1,000 ml PM

    800 ml distilled water

    25 ml 0.5 M Na2HPO4

    25 ml 0.5 M KH2PO4

    10 ml 10% (NH4)2SO4

    1 ml Concentrated base (see below)

    0.1 M Na2S2O3·5H2O

    2 mg/ml p-aminobenzoic acid

    Bring volume to 1,000 ml


    For 1,000 ml Concentrated base

    20 g Nitrilotriacetic acd (NTA-acid free)

    28.9 g MgSO4 anhydrous

    6.7 g CaCl2·2H2O

    18.5 mg (NH4)6Mo7O24·4H2O

    198 mg FeSO4·7H2O

    100 ml Metals 44 (see below)

    Dissolve NTA separately in 600 ml water and neutralize with KOH (14.6 g KOH). Add other components, and dissolve in the order given. Adjust to pH 6.8 before bringing to final volume of 1,000 ml. A precipitate forms when adjusting the pH from the acid side of 6.8 with KOH (need about 100 ml of 1M KOH) but will eventually redissolve with stirring. When the pH is near 6.8, the color of the solution changes from a deep yellow to straw color. Then, filter steriize and store in a glass bottle wrapped in aluminum fold. Store in refrigerator. It is good for at least one year.


    For 1,000 ml Metals 44

    2.5 g (free acid, not sodium salt)

    10.95 g ZnSO4·7H2O

    5 g FeSO4·7H2O

    1.54 g MnSO4·H2O

    392 mg CuSO4·5H2O

    250 mg Co(NO3)2·6H2O

    177 mg Na2B4O7·10H2O

    Add EDTA to 800 ml distilled water with stirring and adjust pH to about 5.0 with 10 M NaOH to get EDTA dissoved. Add the other metals in the order given. Do not add components until the previous one has dissolved completely. Then bring to a final volume of 1,000 ml (the final pH is around 2.4, a clear and lime green solution). Then filter sterilize and store in a glass bottle wrapped in aluminum foil. Can store in refiigerator indefinitely.

  2. Acid-urea gel

    1. Make acid-urea gel solution in a 50 ml centrifuge tube. A recipe for 50 ml of 12% acid-urea gel solution is as follows:

      22.5 g of urea

      1.5 ml of 3 M sodium acetate, pH 5.2

      93.8 µl of 0.5M EDTA, pH 8.0

      18 ml of 30% acrylamide (29:1)

    2. Add RNase-free water and bring the total volume to 45 ml

    3. Add 1 ml of 10% APS

    4. Invert the tube gently to mix

    5. Then add 20 µl of TEMED and invert the tube gently to mix

    6. Pour the solution immediately into the gel cassette

  3. Sodium acetate-EDTA running buffer

    Prepare sodium acetate-EDTA running buffer as follows. The buffer should be pre-chilled to 4 °C before the run.

    100 mM sodium acetate, pH 5.2

    1 mM EDTA

  4. Sodium acetate-EDTA loading buffer

    Prepare sodium acetate-EDTA loading buffer with the following recipe:

    10 ml of RNase-free water

    4.8 g of urea

    20 μl of 0.5 M EDTA, pH 8.0

    33 μl of sodium acetate, pH 5.2

    Pinch of xylene cyanol and bromophenol blue

Acknowledgments

This work was supported by grant W911NF-15-1-0150 to C.S.H. from the U.S. Army Research Office. We thank Dr. Yasuhiro Oda, University of Washington, for his extensive RNA work in R. palustris. We also thank Prof. Michael Ibba, Ohio State University for his excellent advice about tRNA. 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 interest.

References

  1. Bullwinkle, T. J. and Ibba, M. (2016). Translation quality control is critical for bacterial responses to amino acid stress. Proc Natl Acad Sci U S A 113(8): 2252-2257.
  2. Janssen, B. D., Diner, E. J. and Hayes, C. S. (2012). Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis. Methods Mol Biol 905291-309.
  3. 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): .
  4. Kim, M. K. and Harwood, C. S. (1991). Regulation of benzoate-CoA ligase in. Rhodopseudomonas palustris. FEMS Microbiology Letters 83199-203.
  5. https://www.idtdna.com/pages/education/decoded/article/annealing-oligonucleotides.
  6. https://www.nationaldiagnostics.com/electrophoresis/article/run-conditions-denaturing-page.
  7. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/12039672910bul.pdf.

简介

[摘要]转移RNA(tRNA)是遗传密码与蛋白质之间的重要纽带。在翻译过程中,tRNA带有其同源氨基酸,并将其传递至核糖体,因此可作为蛋白质合成的底物。为了分析特定tRNA的电荷状态,纯化总RNA并在酸性尿素凝胶上进行分析。然后将分离的RNA转移到膜上并用目标tRNA的探针进行检测。在这里,我们提出了一种改进的协议来分析α-变形杆菌Rhodopseudomonas palustris中的tRNA充电状态 。与传统方法相比,优化了RNA分离步骤以适合这种生物。另外,非放射性平台用于电泳和RNA印迹。这显着减少了此协议所需的时间和精力。

[背景] tRNA的主要功能是,与其他翻译因素的帮助,以确保mRNA的蛋白质的准确的翻译。氨基酰基-tRNA(带电)将氨基酸带到核糖体中以延长肽段,然后释放不带电荷的tRNA。tRNA的充电状态主要取决于可用资源(即氨基酸)及其被核糖体的消耗量。为了分析细胞tRNA的充电状态,已经开发了使用酸性脲凝胶分离总RNA并通过Northern印迹检测感兴趣的tRNA的方法(Janssen等人,2012; Bullwinkle和Ibba,2016)。最近,我们探索了tRNA充电状态与饥饿细菌存活之间的关系(Yin等人,2019)。该研究中使用的方法,在此进行了详细说明,它优化了RNA纯化步骤,以更好地捕获除大肠杆菌以外的细菌中的tRNA电荷状态。它还利用一个商业上可用的系统来执行分析,而无需使用放射性物质,这对某些研究小组可能会有帮助。

关键字:翻译, tRNA装载, 氨酰转移RNA, 醋酸尿素凝胶, 沼泽红假单胞菌

材料和试剂
 
笔记:
对于该方案,所有材料和试剂都必须不含RNase,这一点至关重要。为此,试剂和材料都是不含RNase的,并且是预制的或易于溶解的,以便尽可能简化程序。只要适用,可使用无RNase的一次性离心管来制备试剂。
DIG试剂有多个试剂盒。此处列出了项目19-23,供读者选择以适合所需的规模。
 
1.离心管,不含RNase(根据需要提供1.5 ml,2 ml,15 ml和50 ml)      
2. QIAzol裂解试剂(Qiagen,目录号:79306)      
3.氯仿,分子生物学等级      
4.异丙醇,分子生物学等级      
5.乙醇,分子生物学等级      
6. UltraPure不含DNase / RNase的蒸馏水(Invitrogen,目录号:10977-023)      
7. TEMED(国家诊断,目录号:EC-503 )      
8.过硫酸铵(国家诊断,目录号:EC-504 )      
9.尿素(National Diagnostics,目录号:EC-605 )      
10. AccuGel 29:1(30%)(National Diagnostics,目录号:EC-851 )   
11. 3 M醋酸钠溶液,pH 4.5(国家诊断,目录号:EC-905)   
12.醋酸钠,3 M溶液,pH 5.2(国家诊断,目录号:EC-906)   
13. EDTA,0.5 M,pH 8.0(Thermo Fisher Scientific,目录号:AM9260G)   
14.二甲苯氰,分子生物学等级   
15.溴酚蓝,分子生物学等级   
16. Tris-HCl溶液,1 M,pH 9.0(Sigma-Aldrich,目录号:T2819-100ML)   
17. Amersham Hybond -XL膜(GE生命科学,目录号:45-001-148)   
18.三- broate -EDTA(TBE)缓冲液,10倍(赛默飞世尔科技,产品目录号:AM9863)   
19. DIG RNA升abeling混合物(西格玛- Aldrich公司,目录号:11277073910)   
20. DIG RN A标签套件(Roche,目录号:11175025910)   
21. DIG系统(罗氏,目录号:12039672910)   
22. DIG Easy Hyb (Sigma - Aldrich,目录号:11603558001)   
23. DIG洗涤和封闭缓冲液组(Sigma - Aldrich,目录号:11585762001)   
24.抗地高辛配基-AP,Fab片段(Sigma - Aldrich,目录号:11093274910)   
25. CDP-Star(Sigma - Aldrich,目录号:11685627001)   
26. RNase Zap(Thermo Fisher,目录号:AM9780)   
27. palustris的生长培养基(PM培养基)(请参阅食谱)   
28.酸性尿素凝胶(请参阅食谱)   
29.乙酸钠-EDTA运行缓冲液(请参见配方)   
30.乙酸钠-EDTA上样缓冲液(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。
 
直径0.1毫米的氧化锆/二氧化硅珠(Bio Spec Products Inc,制造编号:11079101z )
Mini-Beadbeater-24(Bio Spec Products Inc,目录号:112011)
电泳,转移和Northern blot系统适合20厘米高的凝胶
温控台式离心机
用于温控培养或等效培养箱的PCR机
 
小号oftware
 
图像
 
程序
 
从总RNA分离沼泽红假单胞菌
笔记:
为捕获感兴趣的生物选择合适的细胞裂解方法至关重要,以捕获tRNA的天然电荷状态。这里描述的方法基于微珠和打珠器,通常用于我们的研究小组,以从大麦芽孢杆菌和相关α-变形杆菌中分离RNA。我们强烈建议读者针对其感兴趣的生物测试不同的裂解方法(例如,珠子体积与培养基体积之间的比率,细胞跳动循环的次数等)。
关于t RNA充电状态的另一个说明是,正如其他地方所阐述的(Janssen等人,2012;Bullwinkle和Ibba,2016),样品必须始终保持在酸性条件下。整个方案中均使用酸性缓冲液和凝胶系统。
RNA纯化从P rocedure阿可以直接在该协议中的下列步骤中使用。这是因为tRNA的充电状态是根据带电与不带电tRNA的比率计算得出的。因此,DNA和/或蛋白质的轻微污染不会影响此处所述纯化后的最终结果。如果将纯化的RNA用于其他步骤(如RNAseq或体外翻译),则需要进一步纯化。
 
实验前,为每个样品准备一个螺口盖的2 ml离心管。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。
增长沼泽红假单胞菌文化前进水库所要求的实验问题。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。的典型样品含有3-5毫升培养物在阿布斯660〜1.2。通过以4,000 rpm离心在4 °C下收集细胞,并将沉淀保存在-80 °C下。
将每个冷冻细胞沉淀重悬于1 ml QIAzol Lysis Reagent中。用移液器轻轻混合,然后将重悬的样品转移至带有小珠的试管中。此步骤应在冰上进行。
用3500 rpm的打珠器破碎细胞1分钟,然后将样品放在冰上1分钟。重复此循环3次。
将所有样品在室温下放置5分钟。
向每个样品中加入200 µl氯仿。用手剧烈摇动试管15 s。
将试管在室温下放置3分钟。
在4 °C下以12,000 xg离心样品15分钟。小心地将560 µl无色上层水相转移至新的1.5 ml离心管中。
加入0.5毫升异丙醇并涡旋彻底混合。
在4 °C下以12,000 xg离心15分钟。丢弃上清液。
加入1 ml的75%乙醇。在4 °C下以7,500 xg离心5分钟。
完全去除上清液。短暂地在通风橱中风干RNA沉淀(5-10分钟)。
将每个RNA沉淀重新溶于10 mM的乙酸钠,pH 4.5。纯化的RNA可以在-80 °C下保存至少两周。典型的产量是50 µl总RNA 5 µg / µl。该数量可能会因种类,生长状态和采样量等因素而异。
 
碱性pH处理可产生脱酰基的(不带电荷的)tRNA对照
取约10 µg纯化的RNA。直接将0.5 M EDTA和1 M Tris-HCl(pH 9.0)的储备溶液添加到样品中,以将最终缓冲液调整为1 mM EDTA和100 mM Tris-HCl(pH 9.0)。将最终体积调整为50 µl。通过移液混合均匀。
将样品在42 °C下孵育60分钟。
向样品中加入5 µl 3 M乙酸钠,pH 5.2。将缓冲液恢复至pH 5.2不会再次使样品酰化。
向样品中加入137.5 µl冰冷的95%乙醇,然后在冰上孵育10分钟。
在4 °C下以13,000 xg离心10分钟以沉淀RNA。
除去上清液,并用1 ml 70%乙醇洗涤RNA沉淀。
在4 °C下以13,000 xg离心10分钟。
完全去除上清液。根据需要风干RNA沉淀。
用〜30 µl 10 mM乙酸钠,pH 4.5溶解脱酰基的RNA。
 
制备DIG标记的tRNA- Trp探针
DIG RNA标记试剂盒(Roche)在我们的项目中用于生成标记探针,用于后续实验。在适当的情况下,可以替代等效试剂,例如DNase I和T7 RNA聚合酶。其他策略(例如经典的P 32标记)也可以用于此步骤。如果是这样,应相应调整以下实验。作为用于试剂盒DIG的详细协议是可商购的,本节将集中于如何适于它来检测的tRNA色氨酸在沼泽红假单胞菌CGA009。
 
选出R. palustris CGA009 tRNA Trp中的31个核苷酸作为探针的靶标。以下dsDNA已商业化制备(IDT)作为体外转录的模板:
TGAATTG TAATACGACTCACTATAGGG GGTTTTGGAGACCGGTGCTCTACCAATTGAG
根据我们的经验,dsDNA的基本纯化水平应默认为分子生物学级。我们还订购了两个互补的ssDNA,并将它们退火在一起以形成dsDNA(有关更多信息,请参见参考文献5 ),它也能正常工作。
  在上述序列中,阴影部分是T7 RNA聚合酶的启动子,下划线部分是我们在tRNA Trp上靶标的反向互补。在启动子之前添加7 bp的钳位以促进聚合酶结合。必须逐案选择tRNA靶序列,并鼓励读者尝试几种候选序列。
按照DIG RNA标记试剂盒的说明制备反应混合物。以下是我们用于项目的示例(试剂盒中除模板外的所有其他试剂)。可以根据需要放大或缩小音量。
9 µl无RNase的水
4 µl转录(5 x )缓冲液
4 µl DIG标记混合物
2 µl T7聚合酶
1 µl dsDNA模板(10 ng / µl足够)             
将反应混合物在42 °C孵育1.5小时。
对于每个反应,添加2 µl DNaseI,并在37 °C下孵育15分钟。
无需进一步纯化探针。将反应产物稀释并准备用于进一步的步骤。可以使用测试Northern印迹法(例如带有总RNA的点印迹法)来确定用于实际实验的探针的最佳稀释倍数(例如,将探针进行系列稀释10倍,然后进行测试哪种浓度在阅读器的检测系统中产生最佳的信噪比)。
 
酸性尿素聚丙烯酰胺凝胶电泳
建议使用至少20 cm高的电泳系统分析tRNA的充电状态。凝胶/间隔物的厚度无关紧要,只要可以将孔中的尿素充分清除即可。带电和不带电的tRNA之间的差异很小。较小的凝胶可能适合分析某些肽基-tRNA,但通常不提供分析氨酰基-tRNA的分辨率。对于每次运行,必须包括不带电荷的tRNA样品作为阴性对照。
 
用酸性脲凝胶组装电泳系统。添加冷的乙酸钠-EDTA电泳缓冲液,并在20 V 4预运行凝胶30分钟℃下。预运行的目的是提高凝胶的温度。看到公司的网页更多的信息(见参考文献erence 6)。
将样品在72 °C下孵育3分钟。短暂旋转样品,并将其与上样缓冲液按1:1比例混合。对于20厘米高的凝胶,通常每孔上样〜20 µl样品,总共2.5-5 µg总tRNA。所需的最小和最大样品量需要根据具体情况进行测试和确定。
用注射器和针头清洁装载井。这需要在将样品施加到孔之前立即进行。对于酸性脲凝胶,尿素继续在孔中积聚。如果未正确清洁,这可以防止样品沉淀在井底。
加载样品。染料的颜色不应改变。在4 °C下以15 W运行凝胶12小时(对于20厘米高的凝胶)。这是至关重要的,弹出并保持凝胶的操作温度DUR荷兰国际集团的运行,恒功率因此设置以及上述规定的预运行(见参考文献erence 5 F或更多信息)。
 
Northern印迹分析
电泳后,可以使用适合现有系统的任何标准方案将RNA从凝胶转移到膜上。在本例中,我们使用0.5 x TBE作为传输缓冲区。我们在4 °C (20 V过夜或650 mA 30分钟)下进行了标准的湿法转移。两种设置之间未观察到明显差异。然后将膜准备交联并进行Northern印迹。根据我们的经验,暴露于紫外线灯箱中10-15分钟就足够了,并且可以作为起点。
的DIG系统的标准协议(罗氏,见参考文献erence对于m 7矿石信息)瓦特作为用于Northern印迹阻挡,洗涤和检测步骤。简而言之,使用tRNA Trp探针检测膜上的tRNA Trp ,然后与Anti-Digoxigenin-AP和CDP-Star相互作用以产生化学发光信号。任何合适的成像系统,例如胶片或数字成像仪,都可以用来检测和量化结果。
 
数据分析
 
ImageJ用于定量胶片上的图像。对于使用数字成像仪获得的结果,使用随附的软件进行定量。tRNA的充电率计算如下:
 


 
此处简要介绍了tRNA充电凝胶分析。如图1所示;泳道1含有未处理的来自生长的拟南芥的总RNA ,主要含有带电荷的tRNA。相同的样品按照该方案中的描述进行脱酰基处理,并在泳道2中进行分析。待分析的样品在泳道3中。对应于带电和不带电tRNA的谱带通过参考泳道1和2中的谱带确定。使用ImageJ分析各个波段。一个例子,也可在图中找到URE 5d和5e从阴等。(2019)。
 


图1. Northern印迹显示带电和未固定的tRNA Trp的分离
 
菜谱
 
R. palustris生长培养基(PM培养基)
1 ,000毫升PM
800毫升蒸馏水
25毫升0.5 M Na 2 HPO 4
25毫升0.5 M KH 2 PO 4
10毫升10%(NH 4 )2 SO 4
1毫升浓缩碱(见下文)
0.1 M Na 2 S 2 O 3· 5H 2 O
2 mg / ml对氨基苯甲酸
带来体积为1 ,000毫升
 
1 ,000毫升浓碱
20克次氮基ACD (NTA-无酸)
28.9 g无水MgSO 4
6.7克CaCl 2 · 2H 2 O
18.5毫克(NH 4 )6 Mo 7 O 24 · 4H 2 O
198毫克FeSO 4 · 7H 2 O
100 ml金属44(请参阅下文)
将NTA分别溶于600 ml水中,并用KOH(14.6 g KOH)中和。添加其他成分,并按照给定的顺序溶解。使为1最终体积之前调节pH至6.8 ,000毫升。甲沉淀物,用KOH(需要约100ml 1M的KOH)调节时从6.8的酸性侧pH值,但最终将与搅拌再溶解。当pH接近6.8时,溶液的颜色从深黄色变为稻草色。然后,对过滤器进行消毒,然后将其存储在铝折膜包装的玻璃瓶中。存放在冰箱中。至少一年有效。
1 ,000毫升金属44
2.5克(游离酸,不是钠盐)
10.95 g ZnSO 4 · 7H 2 O
5克FeSO 4 · 7H 2 O
1.54 g MnSO 4 · H 2 O
392毫克CuSO 4 · 5H 2 O
250毫克Co(NO 3 )2 · 6H 2 O
177 mg Na 2 B 4 O 7 · 10H 2 O
在搅拌下将EDTA加入800毫升蒸馏水中,并用10 M NaOH将pH调节至约5.0,以除去EDTA 。按给定顺序添加其他金属。在上一个组件完全溶解之前,请勿添加组件。然后带来的1的终体积,000毫升(最终pH为2.4左右,明确和石灰绿的溶液)。然后过滤除菌并储存在铝箔包裹的玻璃瓶中。可以无限期保存在复印机中。
酸性尿素凝胶
在50 ml离心管中制成酸性尿素凝胶溶液。50 ml的12%酸性尿素凝胶溶液的配方如下:
22.5克尿素
1.5 ml 3 M乙酸钠,pH 5.2
93.8 µl 0.5M EDTA,pH 8.0                           
18 ml 30%丙烯酰胺(29:1)                           
加入无RNase的水,使总体积达到45 ml
加入1毫升10%APS
轻轻颠倒试管进行混合
然后加入20 µl TEMED,轻轻颠倒试管进行混合
立即将溶液倒入凝胶盒中
乙酸钠-EDTA运行缓冲液
如下制备乙酸钠-EDTA运行缓冲液。运行前,应将缓冲液预冷至4 °C 。
100 mM醋酸钠,pH 5.2
1毫米EDTA
乙酸钠-EDTA上样缓冲液
使用以下配方准备乙酸钠-EDTA上样缓冲液:
10毫升无RNase的水
4.8克尿素
20 μ升的0.5M EDTA,pH 8.0中的
33 μ升的醋酸钠,pH 5.2
少量的二甲苯腈和溴酚蓝


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小田康弘博士在R. palustris中的广泛RNA工作。我们还要感谢俄亥俄州立大学的Michael Ibba教授对tRNA的出色建议。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
 
TJ Bullwinkle和M.Ibba(2016)。翻译质量控制对于细菌对氨基酸胁迫的反应至关重要。美国国家科学院院刊113(8):2252-2257。              
Janssen,BD,Diner,EJ和Hayes,CS(2012)。通过凝胶电泳分析氨基酰基和肽基tRNA。方法分子生物学905:291-309。              
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)。
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。
https://www.idtdna.com/pages/education/decoded/article/annealing-oligonucleotides。
https://www.nationaldiagnostics.com/electrophoresis/article/run-conditions-denaturing-page。
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/12039672910bul.pdf。
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引用:Yin, L. and Harwood, C. S. (2020). Charging State Analysis of Transfer RNA from an α-proteobacterium. Bio-protocol 10(23): e3834. DOI: 10.21769/BioProtoc.3834.
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