308 



R. B. ROBERTS, R. J. BRITTEN, AND B. J. MCCARTHY 



singer, 19601. A similar conclusion is drawn from X-ray diffraction 

 studies (Zubay and Wilkins, 1960). 



III. Fractionation of Ribosomes 



A. CHROMATOGRAPHY 



Chromatograi)hy on DEAE cellulose columns provides a means for 

 separating protein, S-RNA, ribosomal RNA, and DNA from ribosomes. 

 It is particularly useful in distinguishing the precursors in ribosome 

 synthesis. 



The colunm (1 cm- X 10 cm) is prepared by packing a slurry of 

 DEAE, thoroughly washed with tris buffer (0.01 M, pH 7.6. 0.01 M Mg), 

 at 3-5 psi pressure. The preparation to be analyzed (whole cell juice or 

 ribosome pellet) is adsorbed on the column from this buffer and eluted 

 by 200 ml of the buffer in which there is a linearly increasing concentra- 

 tion of NaCl (0-1 M). 



Under these conditions ribosomes elute at 0.4 M NaCl, S-RNA at 

 0.5 M, and DNA at 0.6 M. Ribosomal RNA (prepared by phenol) 

 remains adsorbed. The ribosomes emerge as 30 and 50S particles with 

 their full complement of protein. If the magnesium concentration is 



E 

 O 



Ci in _ 



o 



(c) 



2.5xlO-3M 



0.40Mn Q50M r 060M 



J i [ 



10 20 



Fraction number 



Fig. 9. Chromatography on DEAE of 70S ribosomes using different concentra- 

 tions of magnesium. Protein content is indicated by S" radioactivity. 



