[962 J.D. W A 1 SON 



1 v( Is which favor the formation of 70s ribosomes from their 30s and 50s 

 sub-units. I ollowing incorporation, those ribosomes possessing nascent poly- 

 peptide chains become less susceptible to breakdown to 30s and 50s ribo- 

 somes. When cell-free extracts (following synthesis) are briefly dialyzed 

 against 10 4 M Mg , about No-90% of the 30s and 50s ribosomes become 

 tree. There remain, however, 10-20% of the original 70s ribosomes and it is 

 upon these i stuck • ribosomes that most ribosomal bound nascent protein is 

 located. This firstly suggests that protein synthesis occurs on 70s ribosomes, 

 not upon free 30s or 50s ribosomes. Secondly, in the commonly studied E. 

 coli extract, only a small ribosomc fraction is functional, Tissieres and Schles- 

 singer named these particles « active ribosomes » and suggested, they con- 

 tained a functional component lacking in other ribosomes. 



Each active nbosome synthesizes on the average between 15,000 and 

 50,000 daltons of protein. This is in the size range of naturally occurring 

 polypeptide chains. Thus while we remained unsatisfied by the small net 

 synthesis, sufficient synthesis occurs to open the possibility that some com- 

 plete protein molecules are made. This encouraged us to look for synthesis 

 of /?-galactosidase. None, however, was then found 29 despite much effort. 



Another important point emerged from these early (1959) incorporation 

 studies with E. coli extracts. Addition of small amounts of purified dcoxy- 

 ribonuclcase decreased protein synthesis to values 20-40% that found in 

 untreated extracts 28 . This was completely unanticipated, for it suggested 

 that high molecular weight DNA functions in the commonly studied bacte- 

 rial extracts. But since a basal synthetic level occurs after DNA is destroyed 

 by deoxyribonuclcase, the DNA itself must not be directly involved in 

 peptide bond formation. Instead, this suggested synthesis of new template 

 RNA upon DNA in untreated extracts. If true, this would raise the possibil- 

 ity, previously not seriously considered by biochemists that the RNA tem- 

 plates themselves might be unstable, and hence a limiting factor in cell-free 

 protein synthesis. 



Metabolic Stability of Ribosomal RNA 



All our early ribosomc experiments had assumed that the ribosomal RNA 

 was the template. Abundant evidence existed that proteins were synthesized 

 on ribosomes and since the template must be RNA, it was natural to assume 

 that it was ribosomal RNA. Under this hypothesis ribosomal RNA was a 



s-120 



