Name: In Vitro Reassociation Assay to Measure the Formation of 80S Ribosomal Particles Using Salt-washed Ribosomal Subunits (Video) | JoVE Journal
Uploaded: 2025-12-16T15:00:00.000+00:00
Duration: 6 min 9 s
Description: Learn an in vitro reassociation assay to measure 80S ribosomal particle formation using salt-washed subunits. This method enables controlled analysis of ribosomal subunit interactions during translation initiation.

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5 minutes

This work was supported by the Estonian Research Council grant (grant number: PRG1741 to T.T.). The research is conducted using the research infrastructure &#34;Experimental Studies and Applications of Cellular Processes - RAKERA&#34; funded by the Estonian Research Council (TARISTU24-TK14). We thank all members of the Chair of Molecular Biology (RiboLab) for helpful discussions. We would like to express our special thanks to Dr. Ivan Kisly for introducing the ribosome subunit purification protocol and the in vitro reassociation protocol in the lab.

1. General materials
<ol>
	<li>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 &#176;C for 20 min. 
	NOTE: At least 1,100 mL of YPD media is required to perform the purification of ribosomal subunits.</li>
	<li>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.</li>
</ol>
2. Purification of budding yeast ribosomal subunits
<ol>
	<li>Growing of yeast cells
	<ol>
		<li>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 &#176;C for 16-20 h.</li>
		<li>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 &#176;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. 
		&#8203;The sucrose gradients as described in steps 2.3.1-2.3.3 can be prepared in advance during the growth of the cells.</li>
	</ol>
	</li>
	<li>Preparation of cell lysate 
	NOTE:The purification of individual ribosomal subunits is adapted from previous publications7,9 with modifications.
	<ol>
		<li>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 &#176;C.</li>
		<li>Prepare approximately 20 pieces of 2 mL screw cap tubes containing 500 &#181;L of glass beads (&#216; 0.25-0.5 mm). Keep at 4 &#176;C.</li>
		<li>Divide the cell culture between centrifuge tubes. Collect the cells by centrifugation in a swinging-bucket rotor at 2,404 &#215; g for 10 min at 4 &#176;C. Discard supernatant.</li>
		<li>Add 200 &#181;L of 1 M DTT and 500 &#181;L 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.</li>
		<li>Transfer suspension into two 50 mL centrifuge tubes. Centrifuge at 2,404 &#215; g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>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 &#181;L per tube).</li>
		<li>Disrupt the cells in a homogenizer at 4 &#176;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.</li>
		<li>Clarify the extracts by centrifugation at 16,060 &#215; g for 5 min at 4 &#176;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.</li>
		<li>Transfer the supernatant into a new 2 mL tube and centrifuge tubes once again at 16,060 &#215; g for 10 min at 4 &#176;C. Collect the lysate in one 50 mL centrifuge tube.</li>
		<li>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.</li>
		<li>To measure an OD, add 2 &#181;L of the obtained lysate to 1,000 &#181;L 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 &#181;L 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.</li>
	</ol>
	</li>
	<li>Separation of 80S ribosomes
	<ol>
		<li>Prepare 10% sucrose solution in Buffer A and 30% sucrose solution in Buffer A as described in Table 4.</li>
		<li>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 &#176;C.</li>
		<li>Balance the prepared sucrose gradients pairwise by carefully removing some of the solution from the surface of the gradient.</li>
		<li>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.</li>
		<li>Ultracentrifuge using a swinging-bucket rotor at 50,339 &#215; g for 16 h (&#969;2t = 2.4 &#215; 1011) at 4 &#176;C.</li>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>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 &#176;C.</li>
		<li>Pool the fractions collected from the gradients in 50 mL centrifuge tube.</li>
		<li>Measure the absorbance at 260 nm using a spectrophotometer and calculate the units of A260 of isolated 80S ribosomes.</li>
		<li>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.</li>
		<li>Ultracentrifuge using a fix-angle rotor at 71,773 &#215; g for 22 h (&#969;2t = 8.0 &#215; 1011) at 4 &#176;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.</li>
	</ol>
	</li>
	<li>Dissociation of ribosomal subunits in high-salt buffer
	<ol>
		<li>Prepare 20 mL of Buffer B as described in Table 3.</li>
		<li>Add 1 mL of ice-cold Buffer B in each centrifuge bottle (from step 2.3.11). Shake the bottles orbitally at 4 &#176;C for 30 min.</li>
		<li>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).</li>
		<li>Balance the prepared sucrose gradients pairwise by carefully removing some of the solution from the surface of the gradient.</li>
		<li>Add 1 mL of ice-cold Buffer B in each tube (from step 2.4.2) and pool them together.</li>
		<li>Measure the absorbance at 260 nm using a spectrophotometer and calculate the units of A260 of isolated ribosomal subunits.</li>
		<li>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).</li>
		<li>Ultracentrifuge using a swinging-bucket rotor at 42,417 &#215; g for 20 h (&#969;2t = 2.8 &#215; 1011) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Concentration of the isolated ribosomal subunits
	<ol>
		<li>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 &#181;L of 1 M DTT and 500 &#181;L of 100 mM PMSF.</li>
		<li>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.</li>
		<li>Measure the absorbance of the collected samples at 260 nm using a spectrophotometer.</li>
		<li>Concentrate the collected subunits using ultrafiltration at 4 &#176;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.</li>
		<li>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.</li>
		<li>Measure the absorbance of the sample at 260 nm using a spectrophotometer. Continue the centrifugation process until the absorbance at A260 reaches 80-150.</li>
		<li>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 &#176;C. The subunits can be stored at -80 &#176;C for up to five years. 
		NOTE:Usually 5x, 10x, 20x, 40x dilutions are used for the measurements of concentration.</li>
	</ol>
	</li>
</ol>
3. Reassociation of eukaryotic ribosomal subunits
NOTE:The in vitro reassociation analysis experiment is adapted from previous publications7,8,9 with modifications.
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Prepare 10% sucrose solutions in Buffer R and 30% sucrose solutions in Buffer R as described in Table 6.</li>
		<li>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.</li>
		<li>Balance the prepared sucrose gradients pairwise by carefully removing some of the solution from the surface of the gradient.</li>
	</ol>
	</li>
	<li>In vitro reassociation reaction of ribosomal subunits 
	NOTE:All solutions used in the in vitro reassociation reaction are at 4 &#176;C and kept on ice.
	<ol>
		<li>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+).</li>
		<li>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 &#181;L 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 &#181;L of 1x Buffer R (200 mM Mg2+) and 206.25 &#181;L of 1x Buffer R (0 mM Mg2+). Add 100 &#181;L 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 &#181;L.</li>
		<li>If using tRNA in the reaction, mix 4 &#181;L of tRNA stock and 96 &#181;L of 1x Buffer R&#160;containing the appropriate magnesium acetate concentration to reach the final concentration of 0.4 mg/mL of tRNA. Prepare the subunit solutions in 100 &#181;L of 1x Buffer R. Ensure that the total volume loaded onto the sucrose gradients remains constant within a rotor.</li>
		<li>Incubate the 40S and the 60S subunit solutions separately at 30 &#176;C for 10 min.</li>
		<li>Mix the 60S subunit solution with the 40S solution and incubate the reaction for an additional 10 min at 30 &#176;C. 
		NOTE:When using tRNA in a reaction, first incubate tRNA solution at 30&#176;C for 10 min, then add tRNA to the 40S subunit solution, subsequently add the 60S subunit solution.</li>
		<li>Terminate the reaction on ice for 5 min.</li>
		<li>Load the reaction carefully to the top of the 10%-30% sucrose gradient (from step 3.1.3).</li>
		<li>Ultracentrifuge using swinging-bucket rotor at 37,368 &#215; g for 20 h (&#969;2t = 2.4 &#215; 1011) at 4 &#176;C.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol><li>Hinnebusch, A. G., Lorsch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanism+of+eukaryotic+translation+initiation%3A+new+insights+and+challenges%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">The mechanism of eukaryotic translation initiation: new insights and challenges.</a> Cold Spring Harb Perspect Biol. 4 (10), a01154(2012).</li>
<li>Fringer, J. M., Acker, M. G., Fekete, C. A., Lorsch, J. R., Dever, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coupled+release+of+eukaryotic+translation+initiation+factors+5B+and+1A+from+80S+ribosomes+following+subunit+joining%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining.</a> Mol Cell Biol. 27 (6), 2384-2397 (2007).</li>
<li>Ben-Shem, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+the+eukaryotic+ribosome+at+3.0+%C3%85+resolution%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The structure of the eukaryotic ribosome at 3.0 Å resolution.</a> Science. 334 (6062), 1524-1529 (2011).</li>
<li>Kisly, I., Tamm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Archaea%2Feukaryote-specific+ribosomal+proteins%3A+guardians+of+a+complex+structure%5BTitle%5D)+AND+%22Comput+Struct+Biotechnol+J%22%5BJournal%5D)">Archaea/eukaryote-specific ribosomal proteins: guardians of a complex structure.</a> Comput Struct Biotechnol J. 21 (1), 1249-1261 (2023).</li>
<li>Schwanhäusser, B., Gossen, M., Dittmar, G., Selbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+analysis+of+cellular+protein+translation+by+pulsed+SILAC%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Global analysis of cellular protein translation by pulsed SILAC.</a> Proteomics. 9 (1), 205-209 (2009).</li>
<li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li>
<li>Kisly, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+functional+role+of+eL19+and+eB12+intersubunit+bridge+in+the+eukaryotic+ribosome%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">The functional role of eL19 and eB12 intersubunit bridge in the eukaryotic ribosome.</a> J Mol Biol. 428 (10 Pt B), 2203-2216 (2016).</li>
<li>Kisly, I., Remme, J., Tamm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosomal+protein+eL24%2C+involved+in+two+intersubunit+bridges%2C+stimulates+translation+initiation+and+elongation%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Ribosomal protein eL24, involved in two intersubunit bridges, stimulates translation initiation and elongation.</a> Nucleic Acids Res. 47 (1), 406-420 (2019).</li>
<li>Tamm, T., Kisly, I., Remme, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+interactions+of+ribosomal+intersubunit+bridges+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Functional interactions of ribosomal intersubunit bridges in Saccharomyces cerevisiae.</a> Genetics. 213 (4), 1329-1339 (2019).</li>
<li>Konikkat, S., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+60S+ribosomal+subunit+assembly+emerging+from+recent+studies+in+yeast%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast.</a> Biochem J. 474 (2), 195-214 (2017).</li>
<li>Steffen, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosome+deficiency+protects+against+ER+stress+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Ribosome deficiency protects against ER stress in Saccharomyces cerevisiae.</a> Genetics. 191 (1), 107-118 (2012).</li>
<li>Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Getting+started+with+yeast%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Getting started with yeast.</a> Methods Enzymol. 350 (1), 3-41 (2002).</li>
<li>Liiv, A., O'Connor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+in+the+intersubunit+bridge+regions+of+23S+rRNA%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Mutations in the intersubunit bridge regions of 23S rRNA.</a> J Biol Chem. 281 (40), 29850-29862 (2006).</li>
<li>Kipper, K., Hetényi, C., Sild, S., Remme, J., Liiv, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosomal+intersubunit+bridge+B2a+is+involved+in+factor-dependent+translation+initiation+and+translational+processivity%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Ribosomal intersubunit bridge B2a is involved in factor-dependent translation initiation and translational processivity.</a> J Mol Biol. 385 (2), 405-422 (2009).</li>
<li>Lilleorg, S., Reier, K., Remme, J., Liiv, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+intersubunit+bridge+B1b+of+the+bacterial+ribosome+facilitates+initiation+of+protein+synthesis+and+maintenance+of+translational+fidelity%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">The intersubunit bridge B1b of the bacterial ribosome facilitates initiation of protein synthesis and maintenance of translational fidelity.</a> J Mol Biol. 429 (7), 1067-1080 (2017).</li>
</ol>

The authors have no conflicts of interest to declare.

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...

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 bridges3,4. 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 yeast7,8,9. 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 &#945;-helix8. Its linker region and C-terminal helix extend from the A-site side of 60S and form the core of the intersubunit bridge eB133,4. 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.

<table><tbody><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Fisher Bioreagents</td><td>BP310-500</td><td></td></tr><tr><td>70 mL centrifuge bottle caps</td><td>Beckman Coulter</td><td>355623</td><td></td></tr><tr><td>Akta UVIS-920-Monitor</td><td>GE Health</td><td>70407</td><td></td></tr><tr><td>Amicon&amp;nbsp;Ultra Centrifugal Filter, 100 kDa MWCO, 15 mL</td><td>Millipore</td><td>UFC9100</td><td></td></tr><tr><td>Bacto Peptone</td><td>Gibco</td><td>211677</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>Gibco</td><td>212720</td><td></td></tr><tr><td>Blue graduated pipette tips, 1000 &amp;micro;L</td><td>TipOne</td><td>S1111-6001</td><td></td></tr><tr><td>Bottle top filtration unit, Funnel only 500 mL</td><td>Starlab</td><td>CC8222-5226</td><td></td></tr><tr><td>C-MAG HS 7 magnetic stir plate</td><td>IKA</td><td>S000</td><td></td></tr><tr><td>Centrifuge Biofuge Fresco</td><td>Heraeus Instruments</td><td>75005510/01</td><td></td></tr><tr><td>Centrifuge bottles 600 mL</td><td>Hettich</td><td>0551</td><td></td></tr><tr><td>Centrifuge Rotina 420R&amp;nbsp;</td><td>Hettich</td><td>4723</td><td></td></tr><tr><td>Centrifuge swing-out rotor&amp;nbsp;</td><td>Hettich</td><td>4784-A+4780+4783</td><td></td></tr><tr><td>D-(+)-glucose monohydrate</td><td>Thermo Scientific</td><td>A11090.36</td><td></td></tr><tr><td>Disposable cuvettes 1.5 mL</td><td>Plastibrand</td><td>759015</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher Bioreagents</td><td>BP172-25</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Thermo Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Fixed-angle rotor 45 Ti</td><td>Beckman Coulter</td><td>339160</td><td></td></tr><tr><td>Glass pearls (&amp;Oslash; 0.25-0.5 mm)</td><td>Carl Roth</td><td>A553.1</td><td></td></tr><tr><td>Gradient maker 100 mL total volume</td><td>Hoefer Scientific Instruments</td><td>SG100</td><td></td></tr><tr><td>Gradient Maker 30 mL total volume&amp;nbsp;</td><td>Hoefer Scientific Instruments</td><td>SG30</td><td></td></tr><tr><td>Heating/cooling dry block CH-100</td><td>BioSan</td><td>BS-010410-CAI</td><td></td></tr><tr><td>Incubator shaker Multitron</td><td>Infors HT</td><td>NC1666704</td><td></td></tr><tr><td>Isopropanol (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;7&lt;/sub&gt;OH)</td><td>Fisher Bioreagents</td><td>BP2618-1</td><td></td></tr><tr><td>Liquid nitrogen (N&lt;sub&gt;2&lt;/sub&gt;)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate (Mg(OAc)&lt;sub&gt;2&lt;/sub&gt; &amp;bull; 4H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>AppliChem</td><td>A7008,0100</td><td></td></tr><tr><td>Micropipette with circular marks, 100 &amp;micro;L</td><td>Marienfeld</td><td>2920116</td><td></td></tr><tr><td>Milli-Q H&lt;sub&gt;2&lt;/sub&gt;O</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Optima XE-90 Ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Orbital shaker</td><td>Sanyo</td><td>Mir-S100C</td><td></td></tr><tr><td>Peristaltic pump PD 5201</td><td>Heidolph</td><td>523-52019-00</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Carl Roth</td><td>6367.1</td><td>PMSF is the only protease inhibitor used in this method.</td></tr><tr><td>Polycarbonate centrifuge bottles, 70 mL, 38 x 102 mm</td><td>Beckman Coulter</td><td>355655</td><td></td></tr><tr><td>Polypropylene centrifuge tubes, 14 x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Polypropylene centrifuge tubes, 25 x 89 mm</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>AppliChem</td><td>A3582,1000</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Lach-Ner</td><td>UN1813</td><td></td></tr><tr><td>Precellys 24 homogenizer</td><td>Bertin Technologies</td><td>P000669-PR-240-A</td><td></td></tr><tr><td>Reaction tubes 3810X 1.5 mL</td><td>Eppendorf</td><td>30125150</td><td></td></tr><tr><td>Safe-Lock tubes 2 mL</td><td>Eppendorf</td><td>&amp;nbsp;0030120094</td><td></td></tr><tr><td>Screw-cap Micro Centrifuge Tube, 2 mL</td><td>VWR</td><td>525-1146</td><td></td></tr><tr><td>Serological pipette 10 mL</td><td>Nerbe plus</td><td>12-461-9208</td><td></td></tr><tr><td>Serological pipette 25 mL</td><td>Nerbe plus</td><td>12-481-9102</td><td></td></tr><tr><td>Spectrophotometer NanoDrop 2000c</td><td>Thermo Scientific</td><td>ND-2000C</td><td></td></tr><tr><td>Spectrophotometer Ultrospec 2000</td><td>Pharmacia Biotech</td><td>CGS 8162.39</td><td></td></tr><tr><td>Sucrose (C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;)</td><td>Carl Roth</td><td>9097.1</td><td></td></tr><tr><td>Swinging-bucket rotor SW28</td><td>Beckman Coulter</td><td>342204</td><td></td></tr><tr><td>Swinging-bucket rotor SW41</td><td>Beckman Coulter</td><td>333790</td><td></td></tr><tr><td>tRNA from E.coli MRE 600</td><td>Roche</td><td>10109541001</td><td></td></tr><tr><td>Yellow pipette tips, 5-200 &amp;micro;L</td><td>Thermo Scientific</td><td>94300120</td><td></td></tr></tbody></table>

in vitro reassociation assay to measure the formation of 80s ribosomal particles using salt-washed ribosomal subunits

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

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.

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...

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.

Ribosomes are molecular machines that are responsible for protein synthesis in all living cells. All ribosomes consist of two subunits. In eukaryotes, the ...

Our research investigates the association of ribosomal subunits during translation initiation elucidating how interactions between subunits regulate efficient protein synthesis in cells universally. This protocol addresses the lack of methods to analyze ribosomal subunit adjoining independent of active translation. To begin, prepare 6, 36 milliliter linear 10 to 30%sucrose gradients with buffer A in polypropylene centrifuge tubes using a gradient mixer.Add 18 milliliters of 30%sucrose solution to the gradient mixer well, positioned further from the exit tubing. Then add 20 milliliters of 10%sucrose solution to the well next to the exit tubing. Ensure that no air bubbles enter the gradient during mixing.Carefully pipette out some of the solution from the surface of each gradient to balance the gradients. Then load up to 150 units of the yeast ribosomal lysate onto the top of each sucrose gradient. Now place the gradients into a swinging bucket rotor and ultracentrifuge at 50, 339 G for 16 hours at four degrees Celsius.Set up the gradient fractionator, or a similar system containing a glass capillary tube, a peristaltic pump, a spectrophotometer, a fraction collector, and a writer. Measure the time required for an air bubble to travel from entering the spectrophotometer to exiting the output tubing. Place the centrifuge tube on ice.From the top of the tube, slide the glass capillary tube straight to the bottom of the gradient. Then pump the gradient through the setup from the bottom to the top while measuring absorbance at 260 nanometers. When the 80S peak appears on the recorded graph, set a timer for this lag time.Begin collecting the gradient only after the lag time has passed, and use the same lag time to end the collection. Collect the 80S peak from all gradients. Prepare six, 11 milliliter linear, 10 to 30%sucrose gradients with buffer R, in polypropylene centrifuge tubes using the gradient mixer.Add 5.5 milliliters of the 30%sucrose solution to the gradient mixer well, positioned further from the exit tubing, and add seven milliliters of the 10%sucrose solution to the well next to the exit tubing. Ensure no bubbles enter the gradient. Balance the prepared sucrose gradients pairwise by carefully removing solution from the surface of each gradient.After terminating the in vitro reassociation reaction, load 300 microliters of the reaction carefully onto the top of the sucrose gradient. Place the gradients into a swinging bucket rotor and ultracentrifuge at 37, 368 G for 20 hours at four degrees Celsius. Using the gradient fractionation system, pump the gradient from the bottom to the top while measuring absorbance at 260 nanometers.Record the absorbance of one tube at a time with the spectrophotometer writer. Centrifugation of the clarified lysate. On a 10%to 30%sucrose gradient, revealed a single peak, corresponding to 80S ribosomal particles in the bottom two thirds of the gradient, along with a peak of low molecular weight nucleic acids near the top.After high salt dissociation of purified 80S particles, centrifugation revealed two peaks corresponding to 40S and 60S ribosomal subunits. In case of successful purification of subunits, a single peak is present in the sample when monitoring the absorbance at 260 nanometers. Under 10 millimolar magnesium acetate conditions, wild-type 40S and 60S subunits reassociated to form a single 80S ribosomal particle peak.The peak of free 40S subunits was visible because 40S subunits were used in a molar excess compared to 60S subunits. When tRNA was added to the 10 millimolar magnesium acetate condition, an additional peak appeared at the top of the gradient, corresponding to excess tRNA. Increasing the magnesium acetate concentration to 20 millimolar, led to the formation of narrower peaks in wild-type reassociation assays.The advantage of this protocol compared to other techniques is the ability to test reassociation of ribosomal subunits under controlled magnesium concentration. The current experimental challenges involve limitations of existing methods that prevent direct analysis of ribosomal subunit joining during translation. The new scientific questions paved by these results involve how ribosomal proteins, RNA, and ligands specifically regulate ribosomal subunit joining.

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Research

JoVE Journal

Biochemistry

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells.  Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed.  As such, this method is the gold standard for examining the regulation of translation.

Eukaryotic Polyribosome Profile Analysis

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Chromatographic Purification of Highly Active Yeast Ribosomes

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely applicable for studying protein-nucleic acid interactions.

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi) is a novel optogenetically activated method developed for labeling and affinity-purifying subcellular populations of ribosomes for downstream sequencing and proteomic analysis.

Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

Learn an in vitro reassociation assay to measure 80S ribosomal particle formation using salt-washed subunits. This method enables controlled analysis of ribosomal subunit interactions during translation initiation.

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

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