Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method

Abstract

One of the major challenges facing researchers working with eukaryotic ribosomes lies in their lability relative to their eubacterial and archael counterparts. In particular, lysis of cells and purification of eukaryotic ribosomes by conventional differential ultracentrifugation methods exposes them for long periods of time to a wide range of co-purifying proteases and nucleases, negatively impacting their structural integrity and functionality. A chromatographic method using a cysteine charged Sulfolink resin was adapted to address these problems. This fast and simple method significantly reduces co-purifying proteolytic and nucleolytic activities, producing good yields of highly biochemically active yeast ribosomes with fewer nicks in their rRNAs. In particular, the chromatographic purification protocol significantly improved the quality of ribosomes isolated from mutant cells. This method is likely applicable to mammalian ribosomes as well. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Figure 1

Methods flowchart. Ribosome purification by the “traditional” method is shown at left. Chromatographic purification using the Cysteine linked sulfolink resin is depicted to the right.

Figure 2

(A) RNA gel electrophoresis analyses. Each lane represents a step in the chromatographic purification process. S30, Spin at 30,000 g; F, Flowthrough; W1, 1^st wash step; W2, 2^nd wash step; W3, 3^rd wash step; E, Elution from resin; CS, Glycerol cushion/supernatant fraction from the overnight spin of the chromatographic purification, CR, final ribosome containing fraction from the chromatographic purification; TS, Glycerol cushion/supernatant fraction from traditional purification; TR, final ribosome containing fraction from the traditional purification. All fractions except CR and TR contain tRNA. One μg of RNA was used for each lane. (B) SDS-PAGE analyses. Each lane represents a step in either the chromatographic or traditional purification process. M, Marker; S30, Spin at 30,000 g; F, Flow through from chromatographic purification; W1, 1^st wash step; W2, 2^nd wash step; W3, 3^rd wash step; E, Elution from the cysteine charged sulfolink resin; CS, Glycerol cushion/supernatant fraction from the overnight spin of the chromatographic purification; CR, final ribosome containing fraction from the chromatographic purification; TS, Glycerol cushion/supernatant fraction from traditional purification; TR, final ribosome containing fraction from the traditional purification. (C) RNase detection assay. A fluor-quench labeled RNA substrate was added to 1 μg of each fraction. Activity was determined by detection of free fluorophore. (D) Protease Activity Assay. A fluor-labeled casein was added to 5 μg of each fraction. Activity was determined by detection of unquenched fluorescein. Activity is represented per μg.

Figure 3

tRNA binding activities and rRNA analysis of ribosomes purified using traditional and chromatographic methods. (A) tRNA saturation binding curves. Ribosomes purified using chromatographic and traditional methods were programmed with PolyU and incubated with increasing amounts of deacylated [³²P] labeled tRNA^Phe. Y-axis denotes the fraction of tRNAs bound per ribosome. X-axis indicates the ratio of input tRNAs to ribosomes. (B) Competition experiment. PolyU programmed, chromatographically purified ribosomes were either first incubated with saturating quantities (500 nM of 80S ribosomes with tRNA^Phe in 25-fold excess) of unlabeled deacylated tRNA^Phe (+tRNA), or buffer alone, and then incubated with 3-fold molar excess of Ac-[¹⁴C]Phe-tRNA^Phe. The ratios of Ac-[¹⁴C]Phe-tRNA^Phe bound per ribosome are indicated. (C) Single site binding isotherms of [¹⁴C]Phe-tRNA to A-sites of ribosomes purified from JD1458 (episomally expressed rRNAs) using chromatographic and traditional methods. (D) Direct reverse transcriptase sequencing of 25S rRNA expressed in JD1458 extracted from ribosomes purified using chromatographic (C) and traditional (T) methods. Read shows sequence from G2945 (bottom) to G2848 (top).