In Vitro Reassociation Assay to Measure the Formation of 80S Ribosomal Particles Using Salt-washed Ribosomal Subunits

Summary

Here, we describe a detailed protocol for the in vitro reassociation of purified, salt-washed eukaryotic ribosomal subunits for the analysis of 80S particle formation. This method is illustrated by analyzing ribosomal subunits isolated from a wild-type strain of Saccharomyces cerevisiae and a mutant strain deficient in the ribosomal protein eL24.

Abstract

Ribosomes are molecular machines that are responsible for protein synthesis in all living cells. All ribosomes consist of two subunits. In eukaryotes, the 40S and 60S subunits interact during translation initiation to form the functional 80S ribosomal particles. These subunits are joined by contacts known as intersubunit bridges. To investigate how damage or mutations affect ribosome functionality, the in vitro reassociation of ribosomal subunits can be employed. In this method, eukaryotic ribosomes are first isolated, then the subunits are dissociated under high-salt condition using sucrose density gradient ultracentrifugation. The purified, salt-washed ribosomal subunits can subsequently be reassociated at different magnesium concentrations to monitor the formation of 80S particles. As an example, the formation of 80S particles using the purified 60S subunits from a Saccharomyces cerevisiae strain lacking the ribosomal protein eL24 is analyzed. Thus, this method enables the investigation of the structural integrity of the purified ribosomal subunits. Additionally, the method enables the evaluation of the roles of ribosomal proteins and rRNA in ribosomal subunit joining outside of the translational context.

Introduction

Protein synthesis has a vital role in all living cells. It is carried out by ribosomes -- ribonucleoprotein complexes composed of RNA molecules and proteins. Ribosomes consist of two subunits, large and small. In eukaryotes, small 40S and large 60S subunit form an 80S particle. Prior to translation initiation, the two ribosomal subunits are separated by the binding of translation initiation factors. During the translation initiation stage, 40S subunit forms a preinitiation complex together with mRNA and several translation initiation factors. This complex scans along the mRNA until the start codon is recognized¹. After the exchange of initiation factors and arrival of 60S, subunit joining is possible². This is achieved by the formation of intersubunit contacts called bridges^3,4. Following the translation initiation stage, a functional ribosomal 80S particle is formed.

Alterations in protein synthesis affect the cell in many aspects. Various methods have been developed to monitor the process. For example, isotopic labelling can be used to quantify protein synthesis by measuring the incorporation of labelled amino acids into newly synthesized proteins⁵. Ribosome profiling, which measures the ribosome occupancy, allows to distinguish between different phases of translation⁶. While these methods can detect the changes in the rate of translation, they do not reveal whether the defect lies within the ribosome or elsewhere in the translation mechanism. The in vitro reassociation assay of purified ribosomal subunits enables investigation of the subunit joining without additional factors. Firstly, functional ribosomal particles are purified and dissociated (Figure 1). Subsequently, the interaction of purified subunits is tested under various conditions. This method allows the assessment of defects in ribosomal proteins and rRNA without translational context. A major advantage of this approach lies in its efficient use of purified ribosomal subunits, requiring only 25-80 pmol of each subunit per reaction. This enables testing of various conditions without the need to repeat the time-consuming purification of ribosomal subunits.

This method has been previously exploited to study the impact of a mutated 60S subunit on the 80S particle formation in budding yeast^7,8,9. The experimental procedures discussed here investigate subunit joining in Saccharomyces cerevisiae strain lacking the ribosomal protein eL24 in comparison to a wild-type strain. eL24 belongs to the large subunit, and it is incorporated into the subunit in the nucleolus during the late stage of maturation¹⁰. eL24 is composed of an N-terminal domain, a linker region, and a C-terminal α-helix⁸. Its linker region and C-terminal helix extend from the A-site side of 60S and form the core of the intersubunit bridge eB13^3,4. eL24 is encoded by paralogous genes in yeast, both of which can be deleted to result in a viable strain¹¹. The exact effect of deleting the full-length and bridge-forming sequence of eL24 on budding yeast has been investigated further⁸. In the absence of eL24, budding yeast cells exhibited a slow growth phenotype and a reduction in global translation. Additionally, the loss of eL24 led to sensitivity to cold and hypersensitivity to antibiotics. In this study, the 60S subunit lacking the full-length eL24 will be reassociated with a wild-type 40S subunit to allow for a more detailed characterization of the effects caused by the loss of eL24 on the subunit joining.

Protocol

1. General materials

1. Prepare Yeast extract-Peptone-Dextrose (YPD)¹² media as described in Table 1. Divide the media into bottles at a volume of up to 400 mL per bottle and sterilize by autoclaving at 115 °C for 20 min.
   NOTE: At least 1,100 mL of YPD media is required to perform the purification of ribosomal subunits.
2. Prepare all the stock solutions required for making buffers as described in Table 2.
   NOTE:The chemicals listed in the Table of Materials can be replaced with identical chemicals of the same grade from different suppliers.
   CAUTION:Dithiothreitol is harmful when swallowed, causes skin and serious eye irritation, and may cause respiratory irritation. Phenylmethylsulfonyl fluoride and potassium hydroxide are harmful if swallowed, and cause severe skin burns and eye damage. Wear personal protective equipment when handling these chemicals. Dispose of chemical waste in accordance with institutional hazardous waste protocols.

2. Purification of budding yeast ribosomal subunits

1. Growing of yeast cells
   1. In the laminar flow cabinet, set up a yeast culture by inoculating a single colony of the desired yeast strain into 50 mL of YPD media. Use a sterile 100 mL Erlenmeyer flask. Grow cells by orbital shaking at 180 revolutions per minute (rpm) at 30 °C for 16-20 h.
   2. Dilute overnight culture in 1 L of YPD with the starting optical density (OD) of 0.3 arbitrary units (AU) at 600 nm (OD₆₀₀). Use a sterile 5 L Erlenmeyer flask. Grow cells by orbital shaking at 180 rpm at 30 °C until the OD₆₀₀ reaches 1.0-1.2 AU.
      NOTE:Growth time varies depending on the generation time of the strain used. The generation time for the wild-type S. cerevisiae strain in YPD is approximately 90 min. When using a mutant strain, it is recommended to determine its specific generation time. The times referred to hereafter are for strains that grow similarly to wild-type.
      ​The sucrose gradients as described in steps 2.3.1-2.3.3 can be prepared in advance during the growth of the cells.
2. Preparation of cell lysate
   NOTE:The purification of individual ribosomal subunits is adapted from previous publications^7,9 with modifications.
   1. Prepare 100 mL of 10x Buffer A without dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) as described in Table 3. Then prepare 200 mL of 1x Buffer A without DTT and PMSF. Keep both at 4 °C.
   2. Prepare approximately 20 pieces of 2 mL screw cap tubes containing 500 µL of glass beads (Ø 0.25-0.5 mm). Keep at 4 °C.
   3. Divide the cell culture between centrifuge tubes. Collect the cells by centrifugation in a swinging-bucket rotor at 2,404 × g for 10 min at 4 °C. Discard supernatant.
   4. Add 200 µL of 1 M DTT and 500 µL of 100 mM PMSF to 100 mL of 1x Buffer A (from step 2.2.1). Divide 50 mL of the 1x Buffer A between centrifuge tubes and resuspend the cell pellet.
   5. Transfer suspension into two 50 mL centrifuge tubes. Centrifuge at 2,404 × g for 5 min at 4 °C. Discard the supernatant.
   6. Resuspend each pellet in 8 mL of ice-cold 1x Buffer A with DTT and PMSF. Divide the suspension between 2 mL screw cap tubes with glass beads (~800 µL per tube).
   7. Disrupt the cells in a homogenizer at 4 °C.
      NOTE:For the homogenizer used here (see Table of Materials), use the program: 3 x 60 s, pause 60 s at 6,000 rpm. Alternatively, a Vortex mixer with multi-socket platform can be used. Use maximum speed and the program: 5 x 60 s, pause 60 s.
   8. Clarify the extracts by centrifugation at 16,060 × g for 5 min at 4 °C. Transfer the supernatant into a new 2 mL tube and repeat the centrifugation.
      NOTE:When transferring the lysate into a new tube, avoid transferring the pellet and glass beads.
   9. Transfer the supernatant into a new 2 mL tube and centrifuge tubes once again at 16,060 × g for 10 min at 4 °C. Collect the lysate in one 50 mL centrifuge tube.
   10. Measure the absorbance at 260 nm using the spectrophotometer to calculate the units of A₂₆₀ of the lysate.
       NOTE:One unit of A₂₆₀ is the amount of nucleic acid contained in 1 mL and producing an OD of 1.
   11. To measure an OD, add 2 µL of the obtained lysate to 1,000 µL of deionized H₂O in a 1.5 mL tube and measure the absorbance at 260 nm using the 1 cm cuvette. The measured A₂₆₀ corresponds to the number of units in 2 µL of lysate. Based on that, calculate the number of units of A₂₆₀ of the lysate.
       NOTE: Alternatively, it is possible to measure the absorbance directly without dilution using a microvolume spectrophotometer.
3. Separation of 80S ribosomes
   1. Prepare 10% sucrose solution in Buffer A and 30% sucrose solution in Buffer A as described in Table 4.
   2. Prepare six 36 mL linear 10%-30% sucrose gradients with Buffer A in polypropylene centrifuge tubes (25 x 89 mm) using the gradient mixer. For each gradient, add 18 mL of 30% sucrose solution to the gradient mixer well positioned further from the exit tubing and 20 mL of 10% sucrose solution to the well next to the exit tubing. Make sure no air bubbles get in the gradient during the mixing.
      NOTE:The additional 2 mL of 10% sucrose solution is used to fill the capillary tubing before each gradient mixing.
      The sucrose gradients must be handled very carefully. The sucrose gradients can be poured beforehand, while the cell culture is growing (step 2.1.2). Keep the prepared gradients undisturbed at 4 °C.
   3. Balance the prepared sucrose gradients pairwise by carefully removing some of the solution from the surface of the gradient.
   4. Load up to 150 units of A₂₆₀ of the lysate (from step 2.2.9) on the top of every 10%-30% sucrose gradient.
      NOTE: Steps 2.3.3. and 2.3.4. need to be done very carefully without disturbing the surface of the sucrose gradient.
   5. Ultracentrifuge using a swinging-bucket rotor at 50,339 × g for 16 h (ω²t = 2.4 × 10¹¹) at 4 °C.
   6. Set up the gradient fractionator or a similar system containing a glass capillary tube, a peristaltic pump, a spectrophotometer, a fraction collector, and writer.
      NOTE:After removing tubes from the ultracentrifuge, keep them undisturbed at 4 °C.
   7. Place the tube on ice. From the top of the tube, slide the glass capillary tube straight to the bottom of the gradient. Pump the gradient through the setup from the bottom to the top and read the absorbance at 260 nm.
   8. Measure the time it takes for an air bubble to travel the setup from entering the spectrophotometer to exiting the output tube. When the 80S peak appears in the graph during recording, set a timer for this lag, and start collecting the gradient only once the lag-time has passed. Use the same lag-time to end the collection. Collect the 80S peak (Figure 2A) from all gradients.
      NOTE:Make sure the pump is turned off when inserting the glass capillary tube in the bottom of the gradient tube. From here onwards, the samples need to always be kept on ice or at 4 °C.
   9. Pool the fractions collected from the gradients in 50 mL centrifuge tube.
   10. Measure the absorbance at 260 nm using a spectrophotometer and calculate the units of A₂₆₀ of isolated 80S ribosomes.
   11. Divide the solution containing the 80S ribosomes between two 70 mL polycarbonate centrifuge bottles. Add ice-cold 1x Buffer A with DTT and PMSF (see steps 2.2.1 and 2.2.4) until at least 2/3 of the bottle is full. Balance the centrifuge bottles.
   12. Ultracentrifuge using a fix-angle rotor at 71,773 × g for 22 h (ω²t = 8.0 × 10¹¹) at 4 °C. Remove the supernatant.
       NOTE:The pellet here looks like a colorless lens. For ease of use, mark the location of the pellet on the bottle.
4. Dissociation of ribosomal subunits in high-salt buffer
   1. Prepare 20 mL of Buffer B as described in Table 3.
   2. Add 1 mL of ice-cold Buffer B in each centrifuge bottle (from step 2.3.11). Shake the bottles orbitally at 4 °C for 30 min.
   3. Meanwhile, prepare 10% sucrose solutions in Buffer B and 25% sucrose solutions in Buffer B as described in Table 4. Thereafter, prepare six 36 mL linear 10%-25% sucrose gradients in Buffer B in polypropylene centrifuge tubes (25 x 89 mm) using the gradient mixer (see step 2.3.2).
   4. Balance the prepared sucrose gradients pairwise by carefully removing some of the solution from the surface of the gradient.
   5. Add 1 mL of ice-cold Buffer B in each tube (from step 2.4.2) and pool them together.
   6. Measure the absorbance at 260 nm using a spectrophotometer and calculate the units of A₂₆₀ of isolated ribosomal subunits.
   7. Load an equal volume of up to 40 units of A₂₆₀ of ribosome solution in Buffer B on the top of the 10%-25% sucrose gradients (from step 2.4.3).
   8. Ultracentrifuge using a swinging-bucket rotor at 42,417 × g for 20 h (ω²t = 2.8 × 10¹¹) at 4 °C.
5. Concentration of the isolated ribosomal subunits
   1. Prepare 100 mL of 1x Buffer C by diluting the 10x stock accordingly (the composition of 10x Buffer C is described in Table 3) and add 200 µL of 1 M DTT and 500 µL of 100 mM PMSF.
   2. Analyze the subunit dissociation using the gradient fractionator by measuring the absorbance at 260 nm from the bottom to the top of the gradient as described in steps 2.3.6-2.3.7. Collect the 60S peak and/or 40S peak from all gradients. The peaks corresponding to the 60S and the 40S subunits are illustrated in Figure 2B.
   3. Measure the absorbance of the collected samples at 260 nm using a spectrophotometer.
   4. Concentrate the collected subunits using ultrafiltration at 4 °C in a micro-concentration device with 100 kDa cut-off regenerated cellulose membrane.
      NOTE:Use the centrifugation conditions as recommended by the manufacturer. If the solution does not fit in the filter at once, centrifuge it, then add more solution and centrifuge again.
   5. Once there is approximately 1 mL of solution on top of the filter, mix the solution with 10 ml of ice-cold 1x Buffer C with DTT and PMSF and repeat centrifugation. Continue this process two more times.
      NOTE: Ribosomal subunits tend to adhere to the filter. Agitate the solution each time 1x Buffer C is added to speed up filtration.
   6. Measure the absorbance of the sample at 260 nm using a spectrophotometer. Continue the centrifugation process until the absorbance at A₂₆₀ reaches 80-150.
   7. To determine the exact units of A₂₆₀ of the concentrated samples, prepare serial dilutions and measure the absorbance of the sample at 260 nm using a spectrophotometer. Divide the dissociated subunits by 5 units of A₂₆₀ in 1.5 mL tubes. Flash freeze in liquid nitrogen and store at -80 °C. The subunits can be stored at -80 °C for up to five years.
      NOTE:Usually 5x, 10x, 20x, 40x dilutions are used for the measurements of concentration.

3. Reassociation of eukaryotic ribosomal subunits

NOTE:The in vitro reassociation analysis experiment is adapted from previous publications^7,8,9 with modifications.

1. Preparation of sucrose gradients
   NOTE:Depending on the experiment carried out, different concentrations of magnesium acetate are used in Buffer R. Use the same concentration of magnesium acetate in the sucrose gradient and in the reaction.
   1. Prepare all 10x and 1x Buffer R with varying magnesium acetate concentrations as described in Table 5. The volume of each buffer is 50 mL.
   2. Prepare 10% sucrose solutions in Buffer R and 30% sucrose solutions in Buffer R as described in Table 6.
   3. Prepare six 11 mL linear 10%-30% sucrose gradients with Buffer R in polypropylene centrifuge tubes (14 x 89 mm) using the gradient mixer. For each gradient, add 5.5 mL of 30% sucrose solution to the gradient mixer well positioned further from the exit tubing and 7.0 mL of 10% sucrose solution to the well next to the exit tubing. Make sure no air bubbles get in the gradient.
      NOTE:The additional 1.5 mL of 10% sucrose solution is used to fill the capillary tubing before each gradient mixing.
   4. Balance the prepared sucrose gradients pairwise by carefully removing some of the solution from the surface of the gradient.
2. In vitro reassociation reaction of ribosomal subunits
   NOTE:All solutions used in the in vitro reassociation reaction are at 4 °C and kept on ice.
   1. Thaw purified 40S and 60S subunits on ice.
      NOTE:The subunits are initially in a buffer containing 5 mM magnesium acetate. Concentrations of magnesium acetate can be varied in the reactions. For that, adjust the amount of added 1x Buffer R (200 mM Mg²⁺).
   2. Since the working concentration of magnesium acetate in the reaction is 10 mM, the initial concentration of magnesium acetate in the purified subunits must be increased from 5 mM to 10 mM. To achieve this, first mix one unit of A₂₆₀ of each subunit separately with 1x Buffer R (5 mM Mg²⁺) to a final volume of 50 µL in 1.5 mL tubes. Next, in a separate 1.5 mL tube, prepare the buffer to change the magnesium acetate concentration for both subunits by mixing 13.75 µL of 1x Buffer R (200 mM Mg²⁺) and 206.25 µL of 1x Buffer R (0 mM Mg²⁺). Add 100 µL of the prepared buffer to each subunit. This will result in both subunits being in 1x Buffer R (10 mM Mg²⁺) at a final volume of 150 µL.
   3. If using tRNA in the reaction, mix 4 µL of tRNA stock and 96 µL of 1x Buffer R containing the appropriate magnesium acetate concentration to reach the final concentration of 0.4 mg/mL of tRNA. Prepare the subunit solutions in 100 µL of 1x Buffer R. Ensure that the total volume loaded onto the sucrose gradients remains constant within a rotor.
   4. Incubate the 40S and the 60S subunit solutions separately at 30 °C for 10 min.
   5. Mix the 60S subunit solution with the 40S solution and incubate the reaction for an additional 10 min at 30 °C.
      NOTE:When using tRNA in a reaction, first incubate tRNA solution at 30°C for 10 min, then add tRNA to the 40S subunit solution, subsequently add the 60S subunit solution.
   6. Terminate the reaction on ice for 5 min.
   7. Load the reaction carefully to the top of the 10%-30% sucrose gradient (from step 3.1.3).
   8. Ultracentrifuge using swinging-bucket rotor at 37,368 × g for 20 h (ω²t = 2.4 × 10¹¹) at 4 °C.
   9. Using the gradient fractionator described in steps 2.3.6-2.3.7, pump the gradient through the setup from the bottom to the top and read the absorbance at 260 nm. Record the absorbance of one tube at a time with the writer of the spectrophotometer.

Results

The in vitro reassociation assay involves two main stages: the purification of ribosomal subunits and the reassociation of ribosomal subunits (Figure 1). Both the 60S and the 40S subunits were purified from a S. cerevisiae wild-type strain. Additionally, the 60S subunit was purified from a strain lacking the ribosomal protein eL24 (ΔeL24). The subunits were purified from the 80S fraction of the cell lysate. Thus, these subunits are capable of forming 80S particles successfu...

Discussion

In vivo, the joining of ribosomal subunits is regulated by several factors, including tRNAs and translation factors that interact with the subunits. The method described here is designed to focus on the impact of changes in ribosomal subunit composition on the formation of 80S particles. The starting point of this method is the purification of individual subunits from functional 80S ribosomes. During this process, ligands associated with the subunits are excluded, and subunits that have not fully undergone the a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Fisher Bioreagents	BP310-500
70 mL centrifuge bottle caps	Beckman Coulter	355623
Akta UVIS-920-Monitor	GE Health	70407
Amicon Ultra Centrifugal Filter, 100 kDa MWCO, 15 mL	Millipore	UFC9100
Bacto Peptone	Gibco	211677
Bacto Yeast Extract	Gibco	212720
Blue graduated pipette tips, 1000 µL	TipOne	S1111-6001
Bottle top filtration unit, Funnel only 500 mL	Starlab	CC8222-5226
C-MAG HS 7 magnetic stir plate	IKA	S000
Centrifuge Biofuge Fresco	Heraeus Instruments	75005510/01
Centrifuge bottles 600 mL	Hettich	0551
Centrifuge Rotina 420R	Hettich	4723
Centrifuge swing-out rotor	Hettich	4784-A+4780+4783
D-(+)-glucose monohydrate	Thermo Scientific	A11090.36
Disposable cuvettes 1.5 mL	Plastibrand	759015
Dithiothreitol (DTT)	Fisher Bioreagents	BP172-25
Falcon 50 mL Conical Centrifuge Tubes	Thermo Scientific	14-432-22
Fixed-angle rotor 45 Ti	Beckman Coulter	339160
Glass pearls (Ø 0.25-0.5 mm)	Carl Roth	A553.1
Gradient maker 100 mL total volume	Hoefer Scientific Instruments	SG100
Gradient Maker 30 mL total volume	Hoefer Scientific Instruments	SG30
Heating/cooling dry block CH-100	BioSan	BS-010410-CAI
Incubator shaker Multitron	Infors HT	NC1666704
Isopropanol (C3H7OH)	Fisher Bioreagents	BP2618-1
Liquid nitrogen (N2)	N/A	N/A
Magnesium acetate tetrahydrate (Mg(OAc)2 • 4H2O)	AppliChem	A7008,0100
Micropipette with circular marks, 100 µL	Marienfeld	2920116
Milli-Q H2O	N/A	N/A
Optima XE-90 Ultracentrifuge	Beckman Coulter	A94471
Orbital shaker	Sanyo	Mir-S100C
Peristaltic pump PD 5201	Heidolph	523-52019-00
Phenylmethylsulfonyl fluoride (PMSF)	Carl Roth	6367.1	PMSF is the only protease inhibitor used in this method.
Polycarbonate centrifuge bottles, 70 mL, 38 x 102 mm	Beckman Coulter	355655
Polypropylene centrifuge tubes, 14 x 89 mm	Beckman Coulter	344059
Polypropylene centrifuge tubes, 25 x 89 mm	Beckman Coulter	326823
Potassium chloride (KCl)	AppliChem	A3582,1000
Potassium hydroxide (KOH)	Lach-Ner	UN1813
Precellys 24 homogenizer	Bertin Technologies	P000669-PR-240-A
Reaction tubes 3810X 1.5 mL	Eppendorf	30125150
Safe-Lock tubes 2 mL	Eppendorf	0030120094
Screw-cap Micro Centrifuge Tube, 2 mL	VWR	525-1146
Serological pipette 10 mL	Nerbe plus	12-461-9208
Serological pipette 25 mL	Nerbe plus	12-481-9102
Spectrophotometer NanoDrop 2000c	Thermo Scientific	ND-2000C
Spectrophotometer Ultrospec 2000	Pharmacia Biotech	CGS 8162.39
Sucrose (C12H22O11)	Carl Roth	9097.1
Swinging-bucket rotor SW28	Beckman Coulter	342204
Swinging-bucket rotor SW41	Beckman Coulter	333790
tRNA from E.coli MRE 600	Roche	10109541001
Yellow pipette tips, 5-200 µL	Thermo Scientific	94300120