Genetic Amino Acid Coding 



441 



pair or wrap about one another, incorpora- 

 tion of phenylalanine is partially or com- 

 pletely reduced. Thus, the synthetic poly- 

 mer is most effective in vitro when single- 

 stranded/ 1 as is messenger RNA in vivo. 



How the presence of different bases in 

 the same synthetic polyribotide affects amino 

 acid incorporation into protein can also be 

 studied. Using polynucleotide phosphoryl- 

 ase, which has riboside diphosphates as sub- 

 strate, polyribotides containing two or more 

 different ribotides can be synthesized in 

 vitro. Nearest-neighbor analysis of the het- 

 eropolymer confirms that the ribotides are 

 actually in a random linear array. The 

 early analyses 7 were greatly expedited be- 

 cause polyphenylalanine is insoluble in the 

 cell-free system. In practice, then, an excess 

 of uridylic acid was used in the synthesis 

 of any mixed polynucleotide to obtain the 

 later-synthesized protein as a precipitate 

 from which the amount and kind of amino 

 acids — in addition to phenylalanine — could 

 be analyzed. Thus, to synthesize polyuri- 

 dylic-adenylic acid, polyuridylic-cytidylic 

 acid, and polyuridylic-guanylic acid, five 

 times as much uridine diphosphate was used 

 as the diphosphates of adenosine, cytidine, 

 or guanosine, respectively. To make mixed 

 polynucleotides containing UAC, UCG, or 

 UGA, ten times as much uridine diphosphate 

 was used as the riboside diphosphates of A, 

 C, or G. 



For example, when a mixed polyribotide 

 containing U and C is added to the cell- 

 free system which is then tested to deter- 

 mine whether an amino acid besides phen- 

 ylalanine is incorporated into protein, pro- 

 line and serine are among the amino acids 

 incorporated. The code letters for these 

 amino acids include, therefore, at least one 

 ribotide of C. In the same way, we can 



8 See M. F. Singer. O. W. Jones, and M. W. 

 Nirenberg (1963). 



7 By S. Ochoa and co-workers, and by M. W. 

 Nirenberg and co-workers. 



also determine the effects of other mixed 

 polyribotides on amino acid incorporation. 

 Some amino acids such as alanine and argi- 

 nine require the use of three different nu- 

 cleotides for coding; thus, the coding ratio 

 (the number of nucleotides required to code 

 one amino acid) is at least three. No amino 

 acid is found which requires the presence 

 of all four types of nucleotides. From these 

 results, it is hypothesized that triplets of nu- 

 cleotides in synthetic messenger RNA are 

 translated into amino acids; that is, a triplet 

 RNA code occurs also in the in vitro stud- 

 ies. 



When the proportions of uridylic acid and 

 cytidylic acid in a mixed polynucleotide are 

 varied, more serine than proline is incorpo- 

 rated when there is an excess of uridylic 

 acid. However, when the excess is cytidylic 

 acid the reverse occurs — more proline than 

 serine is incorporated. In terms of triplets, 

 serine must be specified by 2U 1C and 

 proline by 1U 2C. Note that neither the 

 sequence of nucleotides in the triplet nor 

 the order in which they are read is deter- 

 mined from such results. In other words, 

 although the messenger-RNA triplet code 

 letters are 1U 2C for proline, we cannot 

 say whether the sequence is UCC, CUC, or 

 CCU. (The first and last triplets are dif- 

 ferent, since the single-stranded messenger 

 RNA molecule is translated in one direction 

 only.) 



Starting with ribotides of U and C in the 

 relative frequencies 5:1, the relative fre- 

 quencies of different triplets in the synthe- 

 sized polymer can be predicted. UUU 

 should occur with a frequency of % times % 

 times •;,;, or 1L ' % 1(J . Although three ar- 

 rangements are possible for the code letters 

 2U 1C, any particular sequence should oc- 

 cur with a frequency of % times % times ',-,, 

 or 2 %i 6 ; any one of the three possible 

 arrangements of 1U 2C should occur with 

 a frequency of % times \ ; times %, or 

 % 16 , whereas CCC should occur with a 



