VII. SYNTHESIS OF RNA AND RIBOSOMES 299 



E. RIBOSOMES AS SITE OF PROTEIN SYNTHESIS 



Interest in the biosynthesis of ribosomes stems primarily from the 

 belief that the ribosomes serve as the principal machinery of the cell for 

 protein synthesis. The evidence favoring this view has accumulated over 

 a period of years and is now quite convincing. Cassperson and Brachet 

 observed that RNA was invariably present in high quantity wherever or 

 whenever there was a high rate of synthesis (Cassperson, 1950; Brachet, 

 1950). Caldwell and Hinshelwood showed a quantitative correlation 

 between the quantity of RNA and the growth rate of a wide range of 

 different cells and different conditions (Caldwell and Hinshelwood, 

 1950). Subsequently, when it was found that most of the RNA was 

 located in ribosomes, the same evidence indicated that a high concen- 

 tration of ribosomes was correlated with a high rate of protein synthesis. 



Further evidence appeared in the studies of incorporation of labeled 

 amino acids into protein. In vivo experiments showed that the radioac- 

 tivity of the microsome fraction has a rapid initial rise, whereas that of 

 the soluble proteins has an initial delay suggesting a precursor product 

 relationship (Borsook et al., 1950; Littlefield et al., 1955). In mam- 

 malian tissues the times involved were about 15 minutes and in bacteria 

 the times were about 15 seconds. Furthermore, with the bacteria it was 

 possible to follow the "pulse" of radioactivity with a "chase" of non- 

 radioactive material and to show a transfer of radioactivity from the 

 ribosomes to the soluble protein (McQuillen et al., 1959). 



In vitro experiments showed that ribosomes were an essential com- 

 ponent of any cell-free system capable of incorporating amino acids 

 into protein (Littlefield and Keller, 1957; Tissieres et al., 1960). In these 

 cell-free preparations a large part of the radioactivity incorporated into 

 peptide linkage remained associated with the ribosomes. 



It is now widely accepted that ribosomes furnish the sites of protein 

 synthesis and that the information for the assembly of the amino acids 

 in their proper order is transferred either to the RNA of the ribosomes 

 or to an RNA which is associated with the ribosomes while it acts as 

 template for protein synthesis. 



II. Properties of Ribosomes in Vitro 



A. PREPARATION AND PURIFICATION OF RIBOSOMES 



The preparation of ribosomes begins with the harvesting of the cells. 

 A culture in the desired metabolic condition is rapidly chilled to main- 

 tain that state and centrifuged. The resulting pellet is washed three 

 times to replace the growth medium by a buffer suitable for subsequent 



