The RNA of a dimerized ribosomal structure from hamster cells

45o

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 97241

T H E RNA OF A D I M E R I Z E D RIBOSOMAL S T R U C T U R E FROM H A M S T E R CELLS GEORGE L. E L I C E I R I

Department of Pathology, St. Louis University School of Medicine, St. Lores, 3Io. 63xo 4 (U.N.A.) (Received February 2nd, i972)

SUMMARY

When the ribosomes of Syrian hamster cells are extracted in o.I M K + and 0.02 M Mg 2+ they give rise to dimers, monomers and an intermediate structure. After partial purification of these peaks by sucrose gradient centrifugation, and analysis of their RNA b y polyacrylamide gel electrophoresis, their ratios of 28-S/I8-S RNA indicate that this intermediate structure is made up of one 4o-S and two 6o-S subunits.

Reader and Stanners 1 found that, as a species-specific in vitro phenomenon, in the presence of 0.02 M Mg 2+ and o.I M K + the majority of the free ribosomes from hamster cells sediment in sucrose gradients as dimers. The presence of two intermediate peaks between dimer and monomer ribosomes from Syrian hamster cells (BHKT6a) 2 was later reported a, and they were tentatively assigned the following composition: the faster one made up of one 4o-S and two 6o-S subunits, and the slower one made up of two 6o-S subunits. During an assay for ribosome content in B H K T6a and a series of hamster-mouse hybrid cells derived from it, one intermediate structure was usually resolved in this laboratory 4. The purpose of this work was to learn about the composition of this intermediate peak. Three sparse 84-mm diameter plastic Petri dishes of B H K - T 6 a were incubated for 23 h in Dulbecco's modification of Eagle's medium (Gibco), phosphate-free, 6 ml medium per plate, supplemented with IO % complete bovine serum, with a total of 8 mCi of carrier-free a2Pt (New England Nuclear). The cells were harvested and the ribosome distribution was analyzed by sucrose gradient centrifugation, according to Reader and Stanners z. Briefly, the cells were removed from the plates by incubation with o.i % trypsin for 5 rain at 37°C, washed with phosphate-buffered saline (o.14 M NaC1, 0.003 M KC1, 0.02 M MgC12, o.oi M Tris-HC1, p H 7.4), the pellet was resuspended in H buffer (o.I M KC1, 0.02 M MgC12, o.oi M Tris-HCl, p H 7.4), and incubated in ice for 20 rain. Brij-58 was then added to a final concentration of 0.5 o, /0 and the cells were broken with a Dounce glass homogenizer. After spinning down nuclei and heavy debris for 5 rain at 500 ×g, the supernatant was layered on top of a 29-ml linear gradient of 15-3o % sucrose in H buffer. The gradient was then centrifuged for 16 h at 17 500 rev./min and 4°C, in a Beckmann SW25.I rotor. Fractions of 0.65 ml were collected and 5/,1 aliquots precipitated with 5 % trichloroacetic acid, filtered, and counted by liquid scintillation in toluene. Aliquots of 0.05 ml from the Biochim. Biophys. Acta, 269 (1972) 45o-452

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Fig. I. Sucrose-gradient centrifugation of ribosomes from BFIK-T6a (left panel), resedimentation of fractions from it (3 center panels), and electrophoretic analysis of the RNA isolated from fractions of the latter gradients (3 right panels). The conditions for isotope incorporation, preparation of cytoplasmic extract, sucrose gradient centrifugation, and polyacrylamide gel electrophoresis are described in the text. The arrows in the left panel indicate the fractions from which aliquots were taken for the resedimentations. Similarly, in the center panels the fractions chosen for electrophoretic analysis are identified. D and D', dimer; I and I', intermediate structure; M a n d M', m o n o m e r . I n the right panels, the left peaks are 28-S r R l g A , and the right peaks I 8 - S rlRNA.

fractions indicated in Fig. I were immediately diluted IO times with H buffer and layered on three new similar gradients. The gradients were centrifuged as above, but for 14 h. Again, 0.65 ml fractions were collected and 2o-/,1 aliquots assayed as above. The fractions indicated in the figure were precipitated after addition of nonradioactive, carrier R N A and o.I vol. of 20 ~/o potassium acetate, pH 5.1, by addition of one volume of isopropanol. The resulting ribosomal pellets were then resuspended in 0.5 % sodium dodecyl sulfate and IO % sucrose, and analyzed by polyacrylamide gel electrophoresis as described by Loening ~, with some modifications. Plexiglas tubing of 6-7 m m of internal diameter was used, and the gels were 9 cm long. The composition of the gels was: 2.4 ~o acrylamide, o.12 °/o N,N'-methylene bisacrylamide, 0.04 M Tris-HC1, pH 7.2, 0.02 M sodium acetate, o.ooi M EDTA, IO % glycerol, 0.2 % sodium dodecyl sulfate, 0.08 % N,N,N',N'-tetramethylethylenediamine, and 0.08 ~o ammonium persulfate. The electrophoresis buffer was made up of Tris-HC1, sodium acetate, EDTA, and sodium dodecyl sulfate, in the same concentrations as in the gels. Electrophoresis was carried out at room temperature, at 5 mA per gel, for 60 min. The gels were frozen with dry ice and the upper 3 cm fractionated with a Mickle gel slicer into I-mm-thick slices. Each slice was incubated overnight at room temperature with 0.35 ml toluene and o.05 ml of NCS (AmershamSearle), and counted in a liquid-scintillation counter after addition of io ml of a scintillation mixture containing a 3 : 2 ratio of toluene and ethylene glycol monomethyl ether. Biochim. Biophys. Acta, 269 ( I 9 7 2 ) 4 5 o - 4 5 2

452

G. L. ELICEIRI

In the three right panels of the Fig. i, the ratios of 28-S to I8-S ribosomal RNA (rRNA) are: 2.1 for the monomer, 2.1 for the dimer, and 4.0 for the intermediate peak. These values indicate that this intermediate structure contains two moles of 28-S and I mole of I8-S rRNA, and that therefore, it is most likely made up of two moles of 6o-S and one mole of 4o-S ribosomal subunits. An expected excess of free 4o-S subunits with respect to 6o-S subunits can be seen in the left panel of the figure. As it has been previously reported that molecules of 28-S rRNA, virtually protein-free, can dimerize among themselves 3, ribosome dimerization seems to occur through the 6o-S subunits. The structure studied here might then be two 6o-S subunits attached to each other, with one 4o-S subunit attached to one of the 6o-S particles. Such structure could result from the loss of a 4o-S subunit by a dimer, or from attachment of a 6o-S subunit to a monomer.

ACKNOWLEDGMENT

This work was supported by grant GB-I7546 from the National Science Foundation. REFERENCES I 2 3 4 5

R. G. C. G. U.

V¢. Reader and C. P. Stanners, J. Mol. Biol., 28 (1967) 211. 5larin and J. W. Littlefield, J. Virol., 2 (1968) 69. P. Stanners, G. L. Eliceiri and H. Green, Nature New Biol., 230 (1971) 52. L. Eliceiri, J. Cell Biol., 53 (1972) 177. E. Loening, Biochem. J., lO2 (1967) 251.

Biochim. Biophys. Acta, 269 (1972) 45o-452