[962 |. I). WATSON 



would be no significant accumulation since it would be broken down almost 

 .is tast .is it was made. 



Again the messenger hypothesis was confirmed 38 . The RNA labeled dur- 

 ing pulse exposures was largely attached to 70s and 100s ribosomes in 10 2 M 

 Mg . In low Mg (10 * M), it came offthe ribosomes and sedimented free 

 with an average sedimentation constant of 14s. Base ratio analysis revealed 

 |)NA like RNA molecules in agreement with the expectation that it was 

 produced on very many DNA templates along the bacterial chromosome. 

 Soon afterwards, Hall and Spiegelman 39 formed artificial T2 DNA; T2 

 messenger RNA hybrid molecules and in several laboratories 40 , hybrid mol- 

 ecules were subsequently formed between E. coli DNA and E. coli pulse 

 RNA. The DNA template origin for messenger RNA was thus established 

 beyond doubt. 



The Role of Messenger RNA in Cell-Free Protein Synthesis 



It was then possible to suggest why deoxyribonuclcasc partially inhibits 

 amino acid incorporation in E. coli extracts. The messenger hypothesis 

 prompts the idea that DNA in the extract is a template for messenger RNA. 

 This newly made messenger then attaches to ribosomes where it serves as 

 additional protein templates. Since deoxyribonuclcasc only destroys the ca- 

 pacity to make messenger, it has no effect upon the messenger present at the 

 time of extract formation. Hence, no matter how high the deoxyribonu- 

 clcasc concentration employed, a residual fraction of synthesis will always 

 occur. Experiments by Tissieres and Hopkins 41 in our laboratories and by 

 Berg, Chamberlain, and Wood 42 at Stanford confirmed these ideas. First it 

 was shown that addition of DNA to extracts previously denuded of DNA 

 significantly increased amino acid incorporation. Secondly, RNA synthesis 

 occurs simultaneously with in vitro protein synthesis. This RNA has a DNA 

 like composition, attaches to ribosomes in io~ 2 M Mg ++ , and physically 

 resembles in vivo synthesized messenger RNA. 



Furthermore, Tissieres showed that addition of fractions rich in messenger 

 RNA stimulated in vitro protein synthesis 2-5 fold. More striking results 

 came from Nircnbcrg and Matthaei 43 . They reasoned that in vitro messenger 

 destruction might be the principal cause why cell-free systems stopped syn- 

 thesizing protein. If so, preincubated extracts deficient in natural messenger 

 should respond more to new messenger addition. This way they became 



s-124 



