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

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.