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Ribosome Fractionation in Yeast
酵母核糖体的分离   

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Abstract

This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the polysomes (Figure 1).

Keywords: Ribosome(核糖体), Polysomes(多聚核糖体), Translation(翻译), Gradient centrifugation(梯度离心法), Yeast(酵母)



Figure 1. Ribosome profiles after sucrose gradient centrifugation. Ribosome fractionation was performed in the presence of CHX (black), in the absence of CHX (red) or in the presence of EDTA (green). CHX stabilizes polysomes. In the absence of CHX polysomes are destroyed but 80S is still present. 80S can be completely disrupted in cell extracts by treatment with EDTA.

Materials and Reagents

  1. Glass beads for cells breaking 0.5 mm (Bio Spec Products, catalog number: 110/9105 )
  2. Cycloheximide (CHX) (Sigma-Aldrich, catalog number:  C7698 ) solution 100 mg/ml prepared on ethanol
  3. Bradford reactive (Bio-Rad Laboratories, catalog number:  500-0006 )
  4. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number:  13560400 )
  5. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number:  P7625 ) 100 mM solution prepared on isopropanol
  6. 100% trichloroacetic acid (TCA)  (Fluka, catalog number: 91230 )
  7. Tubes for SW41 rotor Ti (Beckman Coulter, catalog number:  331372 )
  8. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number:  1836170 )
  9. Lysis buffer
  10. Ethanol
  11. Laemmli sample buffer
  12. Bromophenol blue
  13. YPD media (see Recipes)
  14. Buffer A (see Recipes)
  15. 2x buffer B2 times concentrated (see Recipes)
  16. Sucrose gradient (see Recipes)
  17. Foni-inert (see Recipes)

Equipment

  1. Table Centrifuges
  2. Glass bead beater Genie Disruptor (Scientific Industries, catalog number: SI-DD38 )
  3. Ultracentrifuge
  4. Rotor SW41 Ti (Beckman Coulter, catalog number:  331362 )
  5. Density Gradient Fractionation System (ISCO, catalog number: 67-9000-177 )

Procedure

  1. Yeast cultures were grown on YPD or selective media. In the morning dilute night culture till OD600 = 0.15. Grow 100 ml of culture at 30 °C until OD600 = 0.6-0.8.
  2. To keep the polysomes add cycloheximide (CHX) till final concentration 0.1 mg/ml and incubate 10 min on ice.
  3. Spin the cells for 5 min at 4,000 x g and wash with 50 ml of cold water containing 0.1 mg/ml of CHX.
  4. Resuspend the pellets in 1 ml of lysis buffer containing 0.1 mg/ml of CHX, spin. Cells can be frozen and stored at -20 °C.
  5. Work on ice! Resuspend the pellet in 0.4 ml of buffer A, add 0.5 ml of glass beads and disrupt 15 min on the glass bead beater at 4 °C.
  6. To avoid the contamination with cells derby preclean the total extracts. For this transfer the liquid phase into the new tube. Centrifuge 1 min 16,000 x g at 4 °C.
  7. Transfer the supernatant into the new eppendorf tube and centrifuge 10 min 16,000 x g at 4 °C.
  8. Measure the total protein concentrations in the supernatants [= TE (Total Extracts)]. For this dilute the total extracts 1:20 and load 5 and 10 μl to the Bradford sample (0.8 ml of water and 0.2 ml of Bradford reactive).
    Note: Lysate should be fresh, they can not be frozen!
  9. Pour 12 ml gradients (7 to 47% sucrose) using Density Gradient Fractionation System (ISCO) (Figure 2). Gradients are best poured at the night before the centrifugation. Keep the gradients at 4 °C before loading samples. Balance gradient volumes in paired rotor buckets firstly by eye, carefully removing some of top of gradient using a pipette tip if necessary. Check with the balance.


    Figure 2. Preparation of sucrose density gradients for polysome analysis. 6 ml of 7% (marked in blue) and 6 ml of 47% (marked in pink) sucrose solutions are placed into the front and back compartments of a gradient pourer, respectively. A small magnetic flea is used to mix the solution in the front compartment, which is drained slowly using a peristaltic pump to the bottom of the centrifuge tube (approx 10 min per gradient). Care should be taken to minimize the mixing in the centrifuge tube.

  10. Load 0.2 ml of the total extracts containing 2-4 mg of total protein on the top of 7-47% sucrose gradient without disturbing the surface. Note: Do not load more than 0.25 ml of the total extracts!
  11. Centrifuge for 150 min at 220,000 x g (39,000 rpm for rotor SW41) at 4 °C.
  12. Before spin is complete, set up to analyze gradient(s). Standardize absorbance detector with a 1x buffer B. Set baseline according to the Instruction Manual.
    Note: Before putting first gradient on make sure the pump line is filled with foni-inert so that no bubbles can enter sample - this will destroy the gradient! Try briefly raising the needle and running 1 ml of foni-inert into the base cup just prior to first fractionation.
  13. Carefully remove gradients and store at 4 °C without disturbing. Gradients must be fractionated before they start to diffuse. Insert first gradient snugly into top of fractionator [Density Gradient Fractionation System (ISCO)] and raise lower platform to base of tube, and screw-lock. Slowly screw up needle into bottom of gradient tube (Figure 3). Run the pump.


    Figure 3. Fractionation of polysome gradients. Pierce the tube with the needle. Using a peristaltic pump, extrude the gradient from the tube with a foni-inert through a spectrophotometer that monitors the absorbance at 254 nm, and then fractionated into eppendorf tubes. 

  14. End run when first droplets of foni-inert are collected at end of gradient, then put the pump in the reverse mode and pump back most of the foni-inert for reuse. Be careful that no new air bubbles are introduced into pump line and needle during this procedure.
  15. Clean up after fractionating all parts of the gradient fractionation system. Rinse entire system with water, then with 20% ethanol. Clean up bench and equipment. Discard waste solutions and trash.
    Density Gradient Fractionation System (ISCO)
    Isco Type 6 Optical Unit:
    Use 254 nm filter and source screen
    UA-5 Absorbance Detector Settings:
    Chart Speed: 30-60 cm/h
    Sensitivity: 1.0 or 2.0 (A260 Units = Full scale)
    Slope Sensitivity = High
    Pump: Setting 40, clockwise
    Fraction Collector: (if desired)
    Use 1.5 ml microfuge tubes inside larger glass tubes
    Collect 0.4 min drops (0.75 ml)
  16. Take 0.4 ml of fraction (the rest freeze) and add 60 μl of 100% TCA, incubate for 10 min on ice.
  17. Spin at 16,000 x g for 15 min at 4 °C.
  18. Aspirate all traces of TCA and resuspend the pellets in 50 μl of Laemmli sample buffer. 
  19. Load 10 μl on SDS-PAGE
    Note: In case of polysomes disruption, polysomes were dissociated by treatment with 25 mM EDTA added instead of CHX, in the buffer A and in the gradient.

Recipes

  1. YPD media
    2% glucose
    2% bactopeptone
    1% yeast extract
  2. Buffer A
    20 mM Hepes (pH 8.0)
    50 mM KCl
    10 mM MgCl2
    1 % Triton X-100
    1 mM DTT
    1 mM PMSF
    CHX 0.1 μg/ml
    Protease inhibitor cocktail 1 tablet for 10 ml of extract
  3. 2x buffer B2 times concentrated
    40 mM Hepes
    100 mM KCl
    20 mM MgCl2
  4. Sucrose gradient
    7% sucrose on buffer B (1x) + CHX 0.1 mg/ml
    47% sucrose on buffer B (1x) + CHX 0.1 mg/ml
  5. Foni-inert
    50% sucrose on water
    0.01% bromophenol blue

Acknowledgments

This work was supported by grant from Ernst and Lucie Schmidheiny Foundation and Pierre Mercier Foundation awarded to O.O.P. and grants 31003A-120419 and 31003A_135794 of the Swiss National Science Foundation as well as a grant from the Novartis Foundation awarded to M.A.C.

References

  1. Albanese, V., Reissmann, S. and Frydman, J. (2010). A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis. J Cell Biol 189(1): 69-81.
  2. Panasenko, O. O. and Collart, M. A. (2012). Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase. Mol Microbiol 83(3): 640-653.

简介

该协议描述了在蔗糖梯度中的酵母核糖体分级。 在翻译的循环过程中,小(40S)和大(60S)核糖体亚基与mRNA结合以形成80S复合物(单体)。 这种核糖体在翻译延长过程中沿着mRNA移动,然后在终止时解离成40S和60S亚基。 在由一个核糖体延伸期间,另外的核糖体可以在相同的mRNA上启动翻译以形成多核糖体。 每个多核糖体复合物的质量主要由其包含的核糖体的数量决定。 因此,通过蔗糖密度梯度离心,基于mRNA上核糖体的加载,可以对细胞内多核糖体群进行大小分级。 几种化合物有助于维持或破坏多核糖体(图1)。

关键字:核糖体, 多聚核糖体, 翻译, 梯度离心法, 酵母



图1.蔗糖梯度离心后的核糖体谱。在CHX(黑色)存在下,在不存在CHX(红色)或存在EDTA(绿色)的情况下进行核糖体分级分离。 CHX稳定多核糖体。 在没有CHX的情况下,多核糖体被破坏,但80S仍然存在。 80S可以通过用EDTA处理在细胞提取物中完全破坏。

材料和试剂

  1. 用于破碎细胞0.5mm的玻璃珠(Bio Spec Products,目录号:110/9105)
  2. 在乙醇上制备的环己酰亚胺(CHX)(Sigma-Aldrich,目录号:C7698)溶液100mg/ml
  3. Bradford反应性(Bio-Rad Laboratories,目录号:500-0006)
  4. 蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:13560400)
  5. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7625)在异丙醇上制备的100mM溶液
  6. 100%三氯乙酸(TCA) (Fluka,目录号:91230)
  7. SW41转子Ti管(Beckman Coulter,目录号:331372)
  8. 蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:1836170)
  9. 裂解缓冲液
  10. 乙醇
  11. Laemmli样品缓冲区
  12. 溴酚蓝
  13. YPD介质(参见配方)
  14. 缓冲区A(参见配方)
  15. 2x缓冲液B2倍浓缩(见配方)
  16. 蔗糖梯度(参见配方)
  17. Foni惰性(参见配方)

设备

  1. 台离心机
  2. 玻璃珠打浆机Genie Disruptor(Scientific Industries,目录号:SI-DD38)
  3. 超速离心机
  4. Rotor SW41 Ti(Beckman Coulter,目录号:331362)
  5. 密度梯度分级系统(ISCO,目录号:67-9000-177)

程序

  1. 酵母培养物在YPD或选择性培养基上生长。 在早晨稀释夜培养直至OD <600> = 0.15。 在30℃ 下生长100ml培养物,直到OD <600> <0.6-0.8。
  2. 保持多核糖体添加放线菌酮(CHX)直到最终浓度为0.1mg/ml,并在冰上孵育10分钟。
  3. 在4000xg下旋转细胞5分钟,并用50ml含有0.1mg/ml CHX的冷水洗涤。
  4. 将沉淀重悬在1ml含有0.1mg/ml CHX的裂解缓冲液中,旋转。 可以将细胞冷冻并储存在-20℃。
  5. 在冰上工作! 将沉淀重悬于0.4ml缓冲液A中,加入0.5ml玻璃珠,在4℃下在玻璃珠打浆机上破碎15分钟。
  6. 为了避免细胞污染德比预先清除总提取物。为了将液相转移到新管中。在4℃下离心1分钟16,000×g。
  7. 将上清液转移到新的eppendorf管中,并在4℃下离心10分钟16,000×g。
  8. 测量上清液中的总蛋白浓度[= TE(总提取物)]。对于该稀释,总提取物1:20并且向Bradford样品(0.8ml水和0.2ml Bradford反应性)中加载5和10μl。
    注意:裂解液应该是新鲜的,不能冷冻!
  9. 使用密度梯度分馏系统(ISCO)(图2)倾倒12ml梯度(7至47%蔗糖)。梯度最好在离心前的晚上倒入。加样前,保持梯度在4°C。首先通过眼睛平衡成对转子桶中的梯度体积,如果需要,使用移液管尖端小心地移除一些顶部的梯度。请检查天平。



    图2.准备多核糖体分析的蔗糖密度 梯度 > 6 ml的7% 和6ml的47%(以粉红色标记)蔗糖溶液放置在前面 和梯度泵的后室。一个小磁跳蚤 用于混合前隔室中的溶液,其使用缓慢地排出 蠕动泵到离心管的底部(每个梯度约10分钟)。 应小心减少离心管中的混合。

  10. 加载0.2毫升含有2-4毫克总蛋白的总提取物在7-47%蔗糖梯度的顶部,而不干扰表面。 注意:不要加载超过0.25ml的总提取物!
  11. 在4℃下以220,000×g离心150分钟(转子SW41为39,000rpm)。
  12. 在旋转完成之前,设置以分析梯度。使用1x缓冲液B标准吸光度检测器。根据使用手册设置基线。
    注意:在放置第一梯度之前,确保泵管线充满foni惰性,以便没有气泡可以进入样品 - 这将破坏梯度!在第一次分馏之前,尝试短暂地抬起针头,并将1 ml的foni惰性物质倒入底杯中。
  13. 小心移除梯度,并存储在4°C,不打扰。梯度必须在它们开始扩散之前分馏。将第一梯度紧密地插入分馏塔的顶部[密度梯度分馏系统(ISCO)],并将下部平台升至管的底部,并螺旋锁定。将针缓慢拧入梯度管的底部(图3)。运行泵。



    图3.分子的分子 我的渐变。 针。使用蠕动泵,从管中挤出梯度 通过监测254nm处的吸光度的分光光度计, 然后分馏到eppendorf管中。

  14. 当在梯度结束时收集第一雾滴惰性物质时,结束运行,然后将泵置于反向模式,并抽回大部分的惰性气体以便再利用。小心,在此过程中,没有新的气泡被引入泵管线和针。
  15. 在分馏梯度分馏系统的所有部分之后清洗。用水冲洗整个系统,然后用20%乙醇冲洗。清洁工作台和设备。丢弃废液和垃圾。
    密度梯度分馏系统(ISCO)
    Isco 6型光学单元:
    使用254 nm滤镜和源屏幕
    UA-5吸光度检测器设置:
    速度:30-60厘米/小时
    灵敏度:1.0或2.0(A <260单位=满量程)
    斜率敏感度=高
    泵:顺时针设置40,
    馏分收集器:(如果需要)
    在较大的玻璃管内使用1.5 ml微量离心管
    收集0.4分钟滴(0.75ml)
  16. 取0.4ml级分(剩余冷冻)并加入100%TCA的60 μ,在冰上孵育10分钟 。
  17. 在4℃下以16,000×g离心15分钟。
  18. 吸取所有痕量的TCA,并在50 μ l的Laemmli样品缓冲液中重悬沉淀。
  19. 在SDS-PAGE上加载10 μ l 注意:在多核糖体破坏的情况下,通过用25mM EDTA代替CHX在缓冲液A中并以梯度处理来解离多核糖体。

食谱

  1. YPD媒体
    2%葡萄糖 2%细菌用蛋白胨 1%酵母提取物
  2. 缓冲区A
    20mM Hepes(pH 8.0)
    50 mM KCl
    10mM MgCl 2/
    1%Triton X-100 1 mM DTT
    1mM PMSF
    CHX 0.1 μ g/ml
    蛋白酶抑制剂混合物1片用于10ml提取物
  3. 2x缓冲液B2倍浓缩
    40 mM Hepes
    100 mM KCl
    20mM MgCl 2/
  4. 蔗糖梯度
    7%蔗糖的缓冲液B(1x)+ CHX 0.1mg/ml 47%蔗糖的缓冲液B(1x)+ CHX 0.1mg/ml
  5. Foni惰性
    50%蔗糖水溶液
    0.01%溴酚蓝

致谢

这项工作得到Ernst和Lucie Schmidheiny基金会和Pierre Mercier基金会授予O.O.P.的资助。 并授予瑞士国家科学基金会的31003A-120419和31003A_135794以及授予M.A.C.的诺华基金会赠款。

参考文献

  1. Albanese,V.,Reissmann,S。和Frydman,J。(2010)。 促进真核核糖体生物发生的核糖体锚定分子伴侣网络 J Cell Biol 189(1):69-81。
  2. Panasenko,O.O.和Collart,M.A。(2012)。 存在不是和无处不在的 Rps7A 多聚组分取决于Not4 E3连接酶。 Mol Microbiol 83(3):640-653。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Panasenko, O. O. (2012). Ribosome Fractionation in Yeast. Bio-protocol 2(16): e251. DOI: 10.21769/BioProtoc.251.
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Anna Feldman
Tel Aviv University
Dear all,
I have a question: in what fraction of a sucrose gradient (i mean, a percent) I will be able to detect a monosomes?
Many thanks,
Anna
3/8/2015 3:30:54 AM Reply
Olesya Panasenko
Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, Switzerland

Dear Anne,
Peak of monosomes (80S) appears in the middle of 7-47% gradient (if you use my conditions), approximately at 27% of sucrose.

3/9/2015 4:12:50 AM


Anna Feldman
Tel Aviv University

Dear Olesya,
Thanks for your cooperation. May you, please send me a some reference(article) link?
All the best,
Anna

3/9/2015 4:44:19 AM


Olesya Panasenko
Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, Switzerland

I just calculated it by myself :)
You know the length of the gradient - 8.5 cm for 7-47% gradient. The peak of 80S comes exactly in the middle - at 4.2 cm. Gradient is linear. Thus - (7+47)/2 = 27%

3/9/2015 4:57:32 AM


Anna Feldman
Tel Aviv University

Well, but I, simply, write some article and I need the reviewer.

3/9/2015 5:07:17 AM


I feel satisfied after reaindg that one.
9/3/2012 3:50:58 AM Reply