Polysome Profiling without Gradient Makers or Fractionation Systems

Summary

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Abstract

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

Introduction

Ribosomes are mega-Dalton ribonucleoprotein complexes that perform the fundamental process of translating mRNA into proteins. Ribosomes are responsible for carrying out the synthesis of all proteins within a cell. Eukaryotic ribosomes comprise two subunits designated as the small ribosomal subunit (40S) and the large ribosomal subunit (60S) according to their sedimentation coefficients. The fully assembled ribosome is designated as the 80S monosome. Polysomes are groups of ribosomes engaged in translating a single mRNA molecule. Polysome fractionation by sucrose density gradient centrifugation is a powerful method used to create ribosome profiles, identify specific mR....

Protocol

1. Preparation of 7% - 47% sucrose gradients

NOTE: The linear range of the sucrose gradient can be modified to achieve better separation depending on the cell type used. This protocol is optimized for polysome profiles for S. cerevisiae.

1. Prepare stock solutions of 7% and 47% sucrose in sucrose gradient buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl₂, and 1 mM DTT). Filter sterilize the sucrose stock solutions through a 0.22 μm filter and store at 4 °C.
2. Prepare 14 mL of 17%, 27%, and 37% sucrose solutions by dispensing and mixing the 7% and 47% sucrose stock solutions in the manner de....

Results

Three representative polysome profiles are shown in Figure 3. All profiles are from the same yeast strain. A typical polysome profile will have well-resolved peaks for the 40S, 60S, and 80S ribosomal subunits as well as polysomes. The crest of each ribosomal subunit and polysome peak will be apparent on each profile (Figure 3). A representative profile from an automated density fractionation system is shown in Figure 3A. The sucrose.......

Discussion

Here a method to create polysome profiles without the use of expensive automated fractionation systems has been described. The advantage of this method is that it makes polysome profiling accessible to labs that do not have automated fractionation systems. The major disadvantages of this protocol are tedious hand fractionation and reduced sensitivity compared to the dedicated density fractionation system.

This protocol entails careful preparation of sucrose gradients with resolution sufficient.......

Materials

Name	Company	Catalog Number	Comments
Automatic Fractionator	Brandel
Clariostar Multimode Plate Reader	BMG Labtech
Cycloheximide	Sigma Aldrich	C7698
Dithiothreitol	Invitrogen	15508-013
Glass Beads, acid washed	Sigma Aldrich	G8772	425–600 μm
Heparin	Sigma Aldrich	H4784
Magnesium Chloride, 1 M	KD Medical	CAC-5290
Needle, 22 G, Metal Hub	Hamilton Company	7748-08	custom length 9 inches, point style 3
Optima XL-100K Ultracentrifuge	Beckman Coulter
Polypropylene Centrifuge tubes	Beckman Coulter	331372
Polypropylene Test Tube Peg Rack	Fisher Scientific	14-810-54A
Potassium Chloride	Sigma Aldrich	P9541
Qubit 4 Fluorometer	Thermo Fisher Scientific	Q33228
Qubit RNA HS Assay Kit	Thermo Fisher Scientific	Q32855
RNAse Inhibitor	Applied Biosystems	N8080119
Sucrose	Sigma Aldrich	S0389
SW41 Swinging Bucket Rotor Pkg	Beckman Coulter	331336
Syringe, 3 mL	Coviden	888151394
Tris, 1 M,  pH 7.4	KD Medical	RGF-3340
Triton X-100	Sigma Aldrich	X100
UV-Star Microplate, 96 wells	Greiner Bio-One	655801