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CHAPTER 47 



can then be analyzed for the amount and 

 kind of amino acids, besides phenylalanine, 

 which are present. Thus, to synthesize 

 polyuridylic-adenylic acid, polyuridylic- 

 cytidylic acid, and polyuridylic-guanylic acid, 

 five times as much uridine diphosphate is 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 

 is used as the nucleoside diphosphates of A, 

 C, or G. 



For example, a mixed polyribotide com- 

 posed of U and C is added to the cell-free 

 system, which is then tested to determine 

 whether any amino acid besides phenylala- 

 nine is incorporated into protein. With this 

 particular mixture, proline and serine are 

 among the amino acids incorporated. The 

 code letters for these amino acids include, 

 therefore, at least one U and one C. One 

 can also determine, in the same way, the ef- 

 fects of other mixed polyribotides on amino 

 acid incorporation. It is found that some 

 amino acids, such as alanine and arginine, 

 require three different nucleotides to be 

 encoded, so that the coding ratio (the num- 

 ber 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. Were the cod- 

 ing ratio four, there would be 24 different 

 sequences possible in quartets composed of 

 AUGC, some of which might be expected 

 to encode an amino acid.) In view of these 

 results (and of others ^° suggesting a low 

 coding ratio) it is hypothesized that triplets 

 of nucleotides of messenger RNA have 

 amino acid meaning, that is, that we are deal- 

 ing with a triplet RNA code. 



By varying the proportions of uridylic 

 acid and cytidylic acid in a mixed poly- 

 nucleotide, it is discovered that less proline 

 than serine is incorporated when there 

 is an excess of polyuridylic acid. However, 

 10 Obtained by A. Garen (190). 



when there is a relative excess of cytidylic 

 acid in the polymer, the reverse occurs, 

 namely, more proline than serine is incor- 

 porated. In terms of triplets, proline must 

 be specified by UCC and serine as UUC 

 (see Figure 47-3), although the sequence of 

 nucleotides in the triplet and the order in 

 which they are read are still undetermined. 

 In other words, although the messenger RNA 

 triplet code letters are UCC for proline, we 

 cannot say whether the sequence is UCC, 

 cue, or ecu. (The first and last triplets 

 are different, since the single-stranded mes- 

 senger RNA molecule is polarized.) 



Starting with ribotides of U and C in the 

 relative frequencies 5 : 1, we can predict the 

 relative frequencies of different triplets in the 

 synthesized polymer. UUU should occur with 

 a frequency of % X % X %, or i^s^ie. While 

 there are three arrangements possible for the 

 code letters UUC, any particular sequence 

 should occur with a frequency of %X%X )i, 

 or %6; any one of the three possible ar- 

 rangements of UCC should occur with a fre- 

 quency of %X%X %, or %i6, while CCC 

 should occur with a frequency of Kie- These 

 particular sequences are, respectively, in 

 the relative frequencies 125, 25, 5, and 1. 

 Accordingly, if a triplet code is the correct 

 one, then when this particular polyribotide 

 is studied for protein synthesis, one would 

 expect five times more phenylalanine incor- 

 porated than serine, and 25 times more 

 phenylalanine incorporated than proline. 

 Although the results actually obtained " 

 using various synthetic polymers sometimes 

 differ by a factor of two or so from those 

 expected according to the present analysis, 

 the over-all agreement is striking, and offers 

 very strong support for a triplet RNA code. 



(If an amino acid is incorporated at a rate 

 lower than expected, this may be due to the 

 fact that a given nucleotide, U, is in a se- 

 quence which can be read meaningfully in 

 more than two ways. If the sequence is 

 UUt/CCC, sometimes the triplet read may 



