440 



( II \PTER 34 



same triplet modified by the same base sub- 

 stitution, resulting in the incorporation of 

 the same incorrect amino acid into the dif- 

 ferent polypeptide products. These effects 

 can be suppressed by a mutant which mod- 

 ifies the specificity of an enzyme responsi- 

 ble for activating and attaching an amino 

 acid to sRNA. Such a modification may 

 sometimes cause the sRNA to transport an 

 incorrect amino acid to the ribosome carry- 

 ing the abnormal messenger RNA; this 

 amino acid may be the one normally in- 

 corporated at that position in the polypep- 

 tide product. Consequently, mutants which 

 make incorrect messenger RNA may still 

 form the correct protein product, if com- 

 pensated by the additional error of having 

 sRNA carry a specific wrong amino acid. 

 In a limited way, such suppressor mutants 

 cause an alteration in the code for amino 

 acids. 3 



Identification of Codons 



The mechanism of protein synthesis can be 

 studied in vitro by using a suspension of 

 ruptured cells. Such a cell-free system is 

 prepared from E. coli plus the addition of 

 triphosphates of the ribosides of A, G, C, 

 and U as well as all twenty of the amino 

 acids in their L forms. The synthesis of 

 protein can be readily detected if one of the 

 added amino acids is radioactive — valine, 

 for example, which becomes incorporated 

 into protein. This incorporation can be 

 stopped by the addition of DNase, which 

 halts the production of messenger RNA by 

 destroying the DNA. In the absence of 

 new messenger RNA, protein synthesis 

 stops. 



That the DNase effect concerns the pro- 

 duction of messenger RNA is demonstrated 

 by the absence of valine incorporation when 

 sRNA or ribosomal RNA is added to the 



Such mutants are reported by S. Benzer and S. P. 

 Champe and by A. Garen and O. Siddiqi in Proc. 

 Nat. Acad. Sci.. U.S.. 48:1114-1127, 1962. 



system and by the resumption of valine in- 

 corporation when messenger RNA obtained 

 from washed ribosomes is added to the sys- 

 tem. This added messenger RNA can also 

 come from other sources. For example, 

 E. coli extracts can be used to synthesize 

 hemoglobin under the direction of RNA 

 from rabbit reticulocytes, and the RNA of 

 coliphage f2 will stimulate amino acid in- 

 corporation into protein, part of which at 

 least is the coat protein of the phage. 4 



Using such a cell-free system derived 

 from bacteria, we can also study whether 

 the addition of synthetic polyribotides has 

 any effect on protein synthesis. First, a 

 homopolyribotide containing U is added; 

 the polyuridylic acid causes L-phenylalanine 

 to be incorporated into protein. 5 More- 

 over, it is found that: 



1. The protein formed is poly-L-phenyl- 

 alanine 



2. No other amino acid is incorporated 

 in substantial amounts (However, if 

 the Mg+ + concentration is altered or 

 if streptomycin is added, significant 

 amounts of leucine are incorporated. 

 The explanation for this is unknown.) 



3. Phenylalanine linked to sRNA is an 

 intermediate in this process. 



These results surely mean that wherever an 

 appropriate sequence of LPs appears in nor- 

 mal messenger RNA, the protein being syn- 

 thesized will usually incorporate L-phenyl- 

 alanine. This discovery is the first crack 

 in the RNA code; in other words, this is 

 the first determination of a sequence of mes- 

 senger RNA nucleotides which specifies the 

 incorporation of a particular amino acid 

 into protein. 



When the synthetic polyribotide of U is 

 mixed with the synthetic polyribotide of A 

 in a way likely to make the strands base- 



4 See D. Nathans. G. Notani, J. H. Schwartz, and 



N. D. Zinder (1962). 



r See M. W. Nirenberg and J. H. Matthaei (1961). 



