RNA IN THE SYNTHESIS OF PROTEINS 



able to demonstrate a 20-fold increase in protein synthesis following ad- 

 dition of phenol-purified E. coli RNA. Like Tissieres' active fraction, their 

 stimulating fraction scdimented heterogencously arguing against an effect 

 due to cither ribosomal or soluble RNA. More convincing support came 

 when they next added TMV RNA to prcincubated E. coli extracts. Again 

 a 10-20 fold stimulation occurred. Here there could be no confusion with 

 possible ribosomal RNA templates. Even more dramatic 44 was the effect 

 of polyuridylic acid (like TMV RNA single stranded) addition. This specifi- 

 cally directed the incorporation of phenylalanine into polyphenylalanine. 

 With this experiment (June 1961) the messenger concept became a fact. 

 Direct proof then existed that single stranded messenger was the protein 

 template. 



Presence of Messenger RNA in Active Ribosonies 



In in vitro systems ordinarily only 10-20% of E. coli ribosomes contain 

 attached messenger RNA. This first was shown in experiments of Rise- 

 brough 45 who centrifuged extracts of T2 infected cells through a sucrose 

 gradient. Ribosomes containing labeled messenger were found to centrifuge 

 faster than ordinary ribosomes. Similarly, Gilbert 46 showed that these faster 

 scdimenting ribosomes are « active », that is, able to incorporate amino acids 

 into proteins. A fresh cell-free extract was centrifuged through a sucrose 

 gradient. Samples along the gradient were collected and then tested for their 

 ability to make protein. A complete parallel was found between « activity » 

 and the presence of messenger. 



Furthermore, if an extract is centrifuged after it has incorporated amino 

 acids, the nascent protein chains also sediment attached to a small fraction of 

 fast sedimenting ribosomes 45 . These ribosomes still contain messenger RNA. 

 For when the messenger molecules are destroyed by ribonucleasc (ribosomes 

 remain intact in the presence of y amounts of ribonuclease), the ribosomal 

 bound nascent protein sediments as 70s ribosomes. The nascent protein is thus 

 not attached to messenger RNA but must be directly bound to ribosomes. 



Binding ofsRNA to Ribosomes 



Experiments by Schweet 47 and Dintzes 48 show that proteins grow by step- 

 wise addition of individual amino acids beginning at the amino terminal end. 



s-125 



