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

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.

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.

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 successfully in vivo. These purified subunits were then used to analyze the impact of eL24 deficiency on the formation of 80S ribosomal particles in vitro. The results of the subunit purification and reassociation process are presented in the form of sucrose gradient images showing the separation of the particles by ultracentrifugation (Figure 2, Figure 3, and Figure 4).

Ribosomal subunit purification
The purification of ribosomal subunits consists of five steps. Progress in this purification process can be assessed at several checkpoints. First, the cells are lysed, and the resulting lysate is clarified. Typically, 1 L of wild-type cell culture yields 500-1300 units of A260, which are then loaded onto 10%-30% sucrose gradients. After centrifugation, monitoring the absorbance at 260 nm reveals one large peak of 80S ribosomal particles in the bottom two-thirds of the gradient (Protocol step 2.3.8). A peak of low molecular weight nucleic acids found in the lysate can be seen at the top of the gradient (Figure 2A). When using 1 L of wild-type cell culture, approximately 200-800 units of A260 of isolated 80S particles are obtained, which are then used for dissociation of individual subunits in a high-salt buffer. Following the separation of the particles by centrifugation, the sucrose gradients (Protocol step 2.5.2) exhibit two peaks, which correspond to the 60S and the 40S ribosomal subunits (Figure 2B). Furthermore, the absorption at 260 nm of the samples can be used at every step to check the yield of the subunits. By the end of the purification process, the concentration of the individual subunits may vary. Most commonly, the final concentration is maintained at approximately 80-150 units of A260 per mL. Usually, this purification yields 25-75 units of A260 of 40S subunits and 40-100 units of A260 of 60S subunits from 1 L of wild-type cell culture. After purification, the purity of the subunits can be checked by performing an 11 mL of a linear 10%-30% sucrose gradient as described in the reassociation procedure. Load only one unit of A260 of purified subunits onto the gradient without incubation at 30 °C. In case of successful purification of subunits, a single peak is present in the sample when monitoring the absorbance at 260 nm (Figure 3).

Ribosomal subunit reassociation
The ribosomal subunit reassociation is conducted through two steps: reassociating the 40S and 60S subunits and analyzing the formation of 80S ribosomal particle (Figure 1). It is recommended to start with a reassociation assay using wild-type 40S and 60S subunits. This shows whether the purified subunits are capable of joining and whether the conditions for the experiment are appropriate.

The reassociation of the wild-type 40S and 60S subunits has been studied previously7,8,9. It is known that wild-type subunits begin to form 80S particles already in 5 mM magnesium acetate. At 10 mM of magnesium ions, all 60S subunits form 80S particles (Figure 4A). To assess the efficiency of subunit joining, the heights of the 80S and 40S peaks could be analyzed and the relative ratio of these peaks (H80S/H40S) could be calculated. However, such an assessment is only possible if no intermediate particles are formed and the 80S peaks are clearly distinguished. In the reassociation of wild-type subunits at 10 mM magnesium acetate, the formation of a single 80S peak indicates correct subunit reassociation (Figure 4A). The calculated H80S/H40S ratio was ~2.8. The presence of tRNA, in addition to the magnesium ions, promotes the joining of ribosomal subunits. This provides another condition for testing the functionality of purified ribosomal subunits. Excess tRNA in the reaction can be observed as a peak at the top of the gradients (Figure 4B,D,F,H). The assay with wild-type subunits in this experiment demonstrates reassociation and the formation of the 80S particles under all conditions (Figure 4A-D), as previously shown. However, increasing the magnesium ion concentration and adding the tRNA leads to the formation of narrower peaks (Figure 4C,D) with an H80S/H40S ratio of 4.7-6.1. These features are characteristic of stable 80S particles.

Subsequently, the reassociation of the eL24-deficient 60S subunits with wild-type 40S subunits was investigated under various conditions (Figure 4E-H). In the absence of eL24, the 60S subunits displayed a reassociation defect. This was evident at a magnesium acetate concentration of 10 mM, as no 80S particles were formed (Figure 4E). To further characterize the reassociation defect, different concentrations of magnesium ions can be used in the reaction, as magnesium stabilizes the ribosomal particles, supporting their association. Increasing the magnesium acetate concentration or adding tRNA promotes the joining of mutant 60S subunits with wild-type 40S subunits, as evidenced by the formation of intermediate particles (Figure 4F,G). The most efficient formation of 80S particles was achieved by adding tRNA and increasing the magnesium acetate to 20 mM (H80S/H40S ~11) (Figure 4H). Regardless of adding stabilizing factors, some intermediate particles remain, as the 80S formation is less stable than that of wild-type subunits.

In summary, the analysis of in vitro subunit joining indicates that the absence of eL24 impairs the formation of stable 80S particles, which is consistent with the role of eL24 in bridge formation. However, the subunit reassociation defect can be partially rescued by a higher concentration of magnesium ions and tRNA, which highlights the cellular compensatory mechanisms.

Figure 1: A scheme illustrating the setup of a typical experiment. The main steps of this method are highlighted. Scheme created with BioRender. Please click here to view a larger version of this figure.

Figure 2: Purification of ribosomal subunits using sucrose density gradients. (A) Whole-cell extract was prepared from wild-type S. cerevisiae cells growing in YPD media at 30 °C. One hundred and twenty units of A260 were loaded onto the 36 mL of linear 10%-30% sucrose gradient with 10 mM magnesium acetate and centrifuged using a swinging-bucket rotor at 50,339 × g for 16 h (ω2t = 2.4 × 1011) at 4 °C. The absorbance was recorded at 260 nm (A260 nm) and the peak containing 80S particles (indicated by dashed lines) was collected. (B) Purified 80S particles were dissociated in a high-salt buffer. Forty units of A260 were loaded onto the 36 mL of linear 10%-25% sucrose gradient with 5 mM magnesium acetate and 500 mM potassium chloride and centrifuged using a swinging-bucket rotor at 42,417 × g for 20 h (ω2t = 2.8 × 1011) at 4 °C. Sedimentation is from left to right. The absorbance was recorded at 260 nm (A260 nm) and the peaks containing 40S and 60S subunits (indicated by dashed lines) were collected. 40S and 60S subunits and 80S particles are indicated by arrows. Please click here to view a larger version of this figure.

Figure 3: Analysis of the purified ribosomal subunits. One unit of A260 of purified and concentrated ribosomal subunits was loaded onto 11 mL of linear 10%-30% sucrose gradient with 5 mM magnesium acetate and centrifuged using a swinging-bucket rotor at 37,368 × g for 20 h (ω2t = 2.4 × 1011) at 4 °C. Sedimentation is from left to right. The absorbance was recorded at 260 nm (A260 nm). The peaks of (A) wild-type 40S, (B) wild-type 60S, and (C) mutant 60S lacking eL24 are indicated by arrows. Please click here to view a larger version of this figure.

Figure 4: In vitro reassociation assay of purified ribosomal subunits. One unit of A260 of wild-type 40S subunits was co-incubated with (A-D) one unit of A260 of wild-type 60S subunits or (E-H) mutant 60S subunits lacking eL24 in the presence of 10 mM or 20 mM magnesium acetate (10 mM Mg2+; 20 mM Mg2+) for 10 min at 30 °C. Reactions were also performed in the presence of saturating concentrations of deacylated tRNA (10 mM Mg2+ + tRNA; 20 mM Mg2+ + tRNA) to stimulate 80S formation. Ribosomal subunit association was analyzed in 11 mL of linear 10%-30% sucrose gradients with appropriate magnesium acetate concentrations and centrifuged using a swinging-bucket rotor at 37,368 × g for 20 h (ω2t = 2.4 × 1011) at 4 °C. Sedimentation is from left to right. The absorbance was recorded at 260 nm (A260 nm). The peaks of free 40S and 60S ribosomal subunits and 80S particles are indicated by arrows. The intermediate particles are highlighted with asterisks. Please click here to view a larger version of this figure.

Table 1: Yeast extract-Peptone-Dextrose (YPD) media. Please click here to download this Table.

Table 2: Stock solutions for preparation of buffers. Please click here to download this Table.

Table 3: Buffers for ribosomal subunit purification. Please click here to download this Table.

Table 4: Solutions for sucrose gradient preparation for ribosomal subunit purification. Please click here to download this Table.

Table 5: Buffers for ribosomal subunit reassociation. Please click here to download this Table.

Table 6: Solutions for sucrose gradient preparation for ribosomal subunit reassociation. Please click here to download this Table.

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 assembly process are also discarded. The reassociation efficiency of the purified subunits is then tested under controlled conditions and in the presence of different concentrations of magnesium ions.

The method was developed based on similar experiments conducted with bacterial ribosomes. This assay was used to test how the mutant ribosomal RNA or the absence of the bacterial ribosomal protein bL31 affect the subunit joining efficiency^13,14,15. There are several similarities and differences in the purification and in vitro reassociation of bacterial and eukaryotic ribosomal subunits. In both cases, cell lysates are used to isolate the functional ribosomes. The ribosomal subunits are then dissociated. For bacterial ribosomes, a low concentration of magnesium ions is sufficient to promote the dissociation of the subunits. Thus, the subunits are purified from the tight-coupled ribosomes using sucrose gradient centrifugation in the presence of 1 mM magnesium ions. In contrast, dissociation of eukaryotic ribosomal subunits is performed in a high-salt buffer. As with both systems, the subunit reassociation in vitro depends on the concentration of magnesium ions. Reassociation reactions for purified bacterial subunits are performed in buffers containing 6-12 mM of magnesium ions and initiated at 37 °C. For eukaryotic ribosomes, a wider range of magnesium ions is used, and the reaction is carried out at 30 °C. Despite these differences, the method enables the analysis of subunit association in the absence of any other ligands in both bacteria and eukaryotes.

When using this method, it is important to pay close attention to the critical steps. One of the most critical of these is the dissociation and purification of individual subunits. This is particularly important in the case of 40S subunits, as there is a risk of contamination with 60S subunits. To minimize the contamination, the number of units of A₂₆₀ used to separate the dissociated subunits in the sucrose density gradient should be in the range of 30-40. In preparative dissociation, the 60S and 40S peaks must be properly separated from each other and overloading prevents this separation. Contamination by other subunits interferes with the formation of 80S particles, producing inaccurate results that do not reflect the actual impact of the mutant ribosomal component. At the same time, yield must be considered during purification, since experimental design usually requires testing several different conditions.

In the reassociation reaction of this method, 40S subunits are used in a 2-fold molar excess compared to 60S. This is due to two main reasons. Firstly, supplying a surplus of wild-type 40S ensures that the formation of the 80S ribosomal particle is not limited by the amount of 40S subunits. This gives a chance to reassociate for every one of the mutant 60S subunits of this experiment. In case of investigation of mutant 40S subunits, the at least 1.5-fold molar excess of wild-type 60S subunits should be considered. Secondly, the addition of surplus 40S subunits provides a practical advantage in data analysis. Namely, this leads to an additional peak on the gradient image, since not all 40S subunits are incorporated into the 80S particles. These additional peaks can be used to align the gradient images, allowing for more accurate analysis of changes in the reassociation process between different conditions.

This method can be used to analyze the effects of different mutations. To do so, mutant ribosomal subunits are isolated from the budding yeast strain of interest. Mutant ribosomal components (e.g., ribosomal proteins, rRNA) often cause cell growth defects. Therefore, when using mutant yeast strains, it is recommended to determine the growth rate of the cells. Additionally, mutations in ribosomal protein and rRNA genes can reduce the number of ribosomes in cells, resulting in lower yields when purifying subunits. Subunit purification can also be performed by increasing the volume of the cell culture. For example, 2 L of culture can be used for purification instead of 1 L. In this case, for 80S particle separation, 12 gradients rather than the usual six are used. If necessary, the number of sucrose gradients used to dissociate subunits can also be increased to 12. Furthermore, the range of magnesium ions used in the reassociation reaction should correspond to the concentration at which reassociation is successful. Therefore, preliminary experiments should be performed to determine the most relevant magnesium ion concentration range when using this method to characterize different defective subunits.

When analyzing the results of the reassociation reaction, it should be kept in mind that this method is qualitative. In this procedure, all sucrose gradients are prepared manually, resulting in slight differences between them. The results obtained characterize and represent ribosomal subunit behavior in vitro, but do not determine the concentrations of free subunits and 80S particles in the reaction. To ensure reproducibility, it is recommended that a single reassociation condition be repeated at least twice to rule out random deviations. However, if the assessment of the reassociation efficiency is required, the heights of the 80S particle and free 40S subunit peaks could be analyzed, and the relative ratio of these peaks (H_80S/H_40S) could be calculated. Additionally, the widths of the formed 80S peaks could be compared. The effective wild-type subunit joining at a 10 mM magnesium acetate concentration resulted in an H_80S/H_40S ratio ~2.8 (Figure 4A). The addition of tRNA in reaction increased the ratio to 4.6. The subunit reassociation at 20 mM magnesium acetate concentration yielded narrower and higher 80S particle peak and lower free 40S subunit peak (H_80S/H_40S ratio ~5.3) on the gradient image (Figure 4D).

The key strengths of this method are its simplicity, reproducibility and the usage of minimal amounts of ribosomal material. Besides characterizing the joining efficiency of different subunits of S. cerevisiae mutants, this method has potential for various future applications. For instance, different ligands (e.g., RNA molecules, low molecular weight compounds) or protein factors (e.g., mutant translation initiation factors) can be added to the reaction to analyze whether they inhibit or promote the reassociation of subunits. This can be used to analyze both wild-type and mutant ribosomal subunits.