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Analysis of Eukaryotic Translation in Purified and Semipurified Systems

William C. Merrick1,  Jack O. Hensold1

1Case Western Reserve University School of Medicine, Cleveland, Ohio

Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 11.9
DOI: 
10.1002/0471143030.cb1109s08
Online Posting Date: 
May, 2001
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Abstract

Much of the current understanding of the sequential steps involved in translation initiation has been obtained using sucrose gradients to isolate ribosomes and ribosomal subunits, as described here. These purified components are combined with purified translation factors to analyze the formation of intermediates in translation initiation and the roles of the translation factors in vitro.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Identifying Intermediates in the Formation of Protein Synthesis Initiation Complexes
  • Alternate Protocol 1: Formation of 48S Preinitiation Complexes
  • Alternate Protocol 2: Formation of 80S Initiation Complexes
  • Support Protocol 1: Isolation of Ribosomal Subunits Using Preparative Sucrose Gradients
  • Alternate Protocol 3: Monitoring the Position of the Ribosome on Globin mRNA
  • Support Protocol 2: Determination of Relative Sedimentation Coefficients Using Analytical Sucrose Gradients
  • Basic Protocol 2: Analysis of Translation in Cultured Cells
  • Alternate Protocol 4: Purification of Ribosomal Complexes from Cultured Cells for Biochemical Assays
  • Support Protocol 3: Preparation of Yeast Lysates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identifying Intermediates in the Formation of Protein Synthesis Initiation Complexes

 Materials
  • 10% and 40% (w/v) sucrose gradient solutions (see recipe)
  • 1 M Tris×Cl, pH 7.5 (appendix 2A)
  • 1 M KCl
  • 100 mM MgCl2 (see recipe)
  • 100 mM dithiothreitol
  • 100 A260 units/ml AUG
  • 100 mM GTP (see recipe)
  • 150 mM phosphoenolpyruvate
  • 3000 IU/ml pyruvate kinase
  • 10 µM [3H]Met-tRNAi (15 Ci/mmol)
  • 16 µM 40S subunits (see Support Protocol 1)
  • 8 µM eucaryotic initiation factor 2 (eIF2)
  • 15 µM eIF3
  • Aqueous scintillation solution (e.g., Ecoscint, National Diagnostics)
  • 10% (w/v) trichloroacetic acid (TCA) solution, ice cold
  • Acetone
  • SDS sample buffer (see recipe)
  • Gradient maker
  • Refrigerated high-speed centrifuge (e.g., Beckman L7-55)
  • Swinging bucket rotor and appropriate tubes (e.g., Beckman SW56 rotor with 5-ml tubes)
  • UV absorbance detector and chart recorder
  • Syringe or peristaltic pump
  • Fraction collector (e.g., ISCO model 640 gradient fractionator)
  • 90°C oven or heating block
  • Additional reagents and equipment for SDS-PAGE (unit 6.1)

Alternate Protocol 1: Formation of 48S Preinitiation Complexes

 Additional Materials (also see Basic Protocol 1)
  • 1 M KCl
  • 1 M Tris×Cl, pH 7.5 (appendix 2A)
  • 100 mM dithiothreitol

Alternate Protocol 2: Formation of 80S Initiation Complexes

 Additional Materials (also see Basic Protocol 1)
  • 16 µM 60S subunits (see Support Protocol 1)
  • 70 µM eucaryotic initiation factor 1A (eIF1A)
  • 70 µM eIF5A
  • 20 µM eIF5
  • 1 M Tris×Cl, pH 7.5 (appendix 2A)
  • 1 M KCl
  • 100 mM dithiothreitol
  • 100 mM KPO4, pH 8.0
  • Ethyl acetate
  • Scintillation solution (e.g., Econofluor)
  • 13 × 100–mm test tubes
  • Scintillation vials

Support Protocol 1: Isolation of Ribosomal Subunits Using Preparative Sucrose Gradients

 Materials
  • Rabbit reticulocyte lysate (Green Hectares)
  • Standard sucrose solution (see recipe)
  • KCl
  • Subunit buffer (see recipe)
  • 5% and 20% (w/v) sucrose gradient solutions (see recipe)
  • 0.25 M sucrose solution (see recipe)
  • Refrigerated high-speed centrifuge (e.g., Beckman L7-55)
  • Type 35 rotor and 70-ml polycarbonate centrifuge tubes (Beckman)
  • Ti60 or Ti70 rotor and 26-ml polycarbonate centrifuge tubes (Beckman)
  • Gradient maker
  • SW27 rotor and 32-ml polyallomer or cellulose nitrate centrifuge tubes (Beckman)

Alternate Protocol 3: Monitoring the Position of the Ribosome on Globin mRNA

 Additional Materials (also see Basic Protocol 1)
  • mRNA for globin or other gene of interest
  • 32P-end-labeled oligonucleotide primer: e.g., 5¢-TCACCACCAACTTCTTCCAC-3¢ for globin (5000 Ci/mmol), or primer appropriate to gene of interest
  • Micrococcal nuclease–treated rabbit reticulocyte lysate (Promega)
  • 1 M HEPES×KOH, pH 7.5
  • 100 and 500 mM Mg(CH3COOH)2
  • 100 mM dithiothreitol
  • 10 mM anisomycin
  • 10% and 35% (w/v) sucrose gradient solutions (see recipe)
  • 100 mM each dATP, dGTP, dCTP, dTTP
  • AMV reverse transcriptase
  • 1:1 (v/v) phenol/chloroform (appendix 3A)
  • Ethanol
  • 3 M potassium acetate, pH 5.0
  • SW56 rotor and 5-ml polyallomer centrifuge tubes (Beckman)
  • Additional reagents and equipment for DNA sequencing gels (appendix 3A)

Support Protocol 2: Determination of Relative Sedimentation Coefficients Using Analytical Sucrose Gradients

 Additional Materials (also see Basic Protocol 1)
  • 5% and 20% (w/v) sucrose gradient solutions in suitable buffer (e.g., 20 mM Tris×Cl, pH 7.5/100 mM KCl/1 mM dithiothreitol)
  • Protein standards in same buffer:
  •     10 mg/ml ovalbumin (3.55S)
  •     10 mg/ml alddase (7.8S)
  •     10 mg/ml catalase (11.2S)
  •     10 mg/ml -galactosidase (16.1S)
  • Unknown protein

Basic Protocol 2: Analysis of Translation in Cultured Cells

 Materials
  • 10% and 50% (w/v) sucrose gradient solutions (see recipe)
  • Cultured cells or yeast lysate (see Support Protocol 3)
  • PBS (appendix 2A) or other neutral, buffered isotonic solution
  • TMK100 lysis buffer (see recipe), ice cold
  • Tris-buffered, water-saturated phenol: for buffering, use recipe 1 M Tris×Cl, pH 7.5 (appendix 2A)
  • Chloroform
  • Ethanol
  • RNA-loading buffer (see recipe)
  • Gradient maker
  • Refrigerated high-speed centrifuge (e.g., Beckman L7-55)
  • Swinging bucket rotor and appropriate tubes (e.g., Beckman SW28.1 rotor and 17-ml tubes)
  • 15-ml polyethylene tubes with caps
  • UV absorbance detector and chart recorder
  • Syringe or peristaltic pump
  • Fraction collector (e.g., ISCO model 640 gradient fractionator)
  • Water bath or heating block at 65°C
  • Additional reagents and equipment for analyzing RNA on agarose or acrylamide gels and for northern blotting (appendix 3A)

NOTE: Experiments involving RNA require careful precautions to prevent contamination and RNA degradation (see appendix 2A).

NOTE: All procedures are carried out on ice with solutions that have been precooled to 4°C.


Alternate Protocol 4: Purification of Ribosomal Complexes from Cultured Cells for Biochemical Assays

 Additional Materials (also see Basic Protocol 2)
  • Cultured cell lysate (Basic Protocol 2) or yeast lysate (Support Protocol 3)
  • 25% sucrose gradient solution (buffered as for 10% and 50% solutions in Basic Protocol 2)
  • TMK100 lysis buffer (see recipe) without detergent
  • 22% (w/v) sucrose solution (see recipe)
  • Swinging bucket rotor and tubes (e.g., Beckman SW50.1 rotor and 5-ml tubes)

Support Protocol 3: Preparation of Yeast Lysates

 Materials
  • Yeast culture
  • Breaking buffer (see recipe)
  • Dry acid-washed glass beads (425- to 600-µm; Sigma)
  • 1 mg/ml cyclohexamide
  • Spectrophotometer
  • Additional reagents and equipment for growing yeast (unit 1.6)
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Figures

  • Figure 11.9.1
    Methods for collecting sucrose gradients. In the example shown in (A), the top of the centrifuge tube is fitted into an adaptor that fits into the UV detector. A dense sucrose solution (i.e., >50% w/v) is introduced into the bottom of the centrifuge tube with the aid of a tube-piercing apparatus that seals the bottom of the tube. As the heavy sucrose is introduced, the gradient is displaced upwards through the UV detector and then to a fraction collector. In the example shown in (B), a thin metal tube is carefully inserted to the bottom of the centrifuge tube. The sample is pumped out of the bottom of the tube, through the UV detector, and then to the fraction collector.

    View Image
  • Figure 11.9.2
    Reactivity of Met-tRNAi bound to 40S and 80S initiation complexes.In this experiment, eIF2, eIF3, AUG, GTP, [3H]Met-tRNAi, and 40S subunits were incubated 15 min at 30°C in a 100-µl volume. After incubation, the reaction was layered onto a 10% to 35% sucrose gradient (buffered in 20 mM Tris×Cl, pH 7.5, 100 mM KCl, and 5 mM MgCl2). After centrifugation at 55,000 rpm at 4°C for 140 minutes, the gradients were fractionated (250-µl fractions) by displacement with 40% sucrose (Fig. 11.9.1A). Aliquots were sampled for radioactivity (closed circles) and for reactivity of Met-tRNAi with puromycin (expressed as a percentage of cpm bound Met-tRNAi, shaded bars). (A) Also included in the incubation were eIF5 and 60S subunits. (B) [14C]eIF5 (open circles) was present in the incubation; 60S subunits were added to test for reactivity with puromycin. (C) GDPNP replaced GTP in the incubation; eIF5 and 60S subunits were added to test for reactivity with puromycin. (Adapted from Peterson et al., 1979b.) This figure represents an experiment where different complexes are resolved by sucrose gradients. Met-tRNAi, present in all experiments, was monitored by liquid scintillation spectrometry. The ability of bound Met-tRNAi to form methionyl-puromycin was determined separately. Previous studies had shown that formation of methionyl-puromycin required 40S and 60S subunits, AUG, eIF2, eIF3, eIF5, and Met-tRNAi. eIF1A and eIF5A stimulate this process as well.

    View Image
  • Figure 11.9.3
    Sedimentation of ribosomes from erythroleukemic cells in 10% to 50% and 10% to 25ucrose gradients. Erythroleukemic cells in log phase of growth were lysed as described in the text, and post-nuclear supernatants were loaded onto 10% to 25% sucrose gradients. The gradients were collected with an ISCO model 640 gradient fractionator by displacement from the bottom with 60% sucrose into a flow cell (Fig. 11.9.1A). UV absorbance was continuously monitored during the collection. The gradients contained 20 mM HEPES×KOH, pH 7.4, 5 mM MgCl2, 100 mM KCl, and 2 mM dithiothreitol. In the experiment shown in the far right panel, 10 mM EDTA was added to the post-nuclear supernatants and to the gradients. The positions of sedimentation of the 40S, 60S, and 80S ribosomes are indicated by arrows above the figures. These assignments were confirmed by analysis of the rRNA content of the inclusive fractions. The direction of sedimentation is from right to left.

    View Image
  • Figure 11.9.4
    Serum-starved 3T3 cells and erythroleukemic cells exposed to inducers of differentiation demonstrate evidence of a decrease in the rate of translation initiation. Murine erythroleukemic (MEL) cells were grown for 9 hr in the presence or absence of the inducer of differentiation A23187 (0.75 µg/ml). BALB/c 3T3 cells were grown to confluence, and then either (1) replated in fresh medium containing 10% calf serum (control), or (2) washed with PBS, refed with medium containing 0.5% calf serum (0.5% CS), and incubated for an additional 18 hr. Cell lysates were prepared for analysis on 10% to 50% sucrose gradients as described in the text. Gradients were collected with an ISCO 640 gradient fractionator with continuous monitoring of the UV absorbance at 254 nm (Fig. 11.9.1A). Positions of 40S, 60S, and 80S ribosomal peaks are indicated by arrows above each tracing. The direction of sedimentation is from right to left.

    View Image

Literature Cited

Literature Cited
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    Hensold, J., Barth-Baus, D. and Stratton, C. 1996. Inducers of erythroleukemic differentiation cause mRNAs that lack poly(A)-binding protein to accumulate in translationally inactive, salt-labile 80S ribosomal complexes. J. Biol. Chem. 271:23246-23254.
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    Martin, T. 1973. A simple general method to determine the proportion of active ribosomes in eukaryotic cells. Exp. Cell. Res. 80:496-498.
    Martin, T. and Hartwell, L. 1970. Resistance of active yeast ribosomes to dissociation by KCl. J. Biol. Chem. 245:1504-1508.
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    Merrick, W.C. 1979a. Purification of protein synthesis initiation factors from rabbit reticulocytes. Methods Enzymol. 60:101-108.
    Merrick, W.C. 1979b. Assays for eukaryotic protein synthesis. Methods Enzymol. 60:108-123.
    Merrick, W. 1992. Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56:291-315.
    Merrick, W.C. Anderson, W.F. and 1975. Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes. J. Biol. Chem. 250:1197-1206.
    Moritz, M., Pulaski, B. and Woolford, J. 1991. Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16. Mol. Cell Biol. 11:5681-5692.
    Nielsen, P. and Trachsel, H. 1988. The mouse protein synthesis initiation factor 4A gene family includes two related functional genes which are differentially expressed. EMBO J. 7:2097-2105.
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    Peterson, D.T., Safer, B. and Merrick, W.C. 1979b. Role of eukaryotic initiation factor 5 in the formation of 80S initiation complexes. J. Biol. Chem. 254:7730-7735.
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