442 



( II AIM I K 34 



frequency of 54ie« These particular se- 

 quences are. respectively, in the relative 

 frequencies 125:25:5:1. Consequently, it 



a triplet code is the correct one. studies 

 of this particular polyribotide for protein 

 synthesis, should reveal incorporation of 

 five times more phenylalanine than serine 

 and twenty-five times more phenylalanine 

 than proline. Although the results obtained 

 using various synthetic polymers sometimes 

 differ by a factor of two or so from those 

 presently expected, the overall agreement is 

 excellent and offers very strong support for 

 a triplet RNA code. The existence of a 

 triplet code is also supported by the finding 

 that the messenger RNA is approximately 

 450 ribotides long B while the a or ft chain 

 which it specifies in hemoglobin is 150 or 

 so amino acids long. 



All the synthetic polyribotides tested thus 

 far for messenger RNA activity in protein 

 synthesis contain an excess of U for tech- 

 nical reasons. As mentioned earlier, the 

 protein product is mainly polyphenylala- 

 nine, insoluble in the cell-free system and 

 therefore readily collected and quantitatively 

 analyzed for phenylalanine as well as other 

 amino acids. Such studies reveal triplet code 

 letters for nineteen amino acids. For exam- 

 ple, three triplets code for leucine — 1A 2U, 

 1C 2U, and 1G 2U — demonstrating, as ex- 

 pected from our previous discussion, that 

 in vitro, at least, the code is degenerate. 

 Degeneracy also occurs for asparagine 

 which has 2 A 1U and 1C 1A 1U as co- 

 dons, and isoleucine with codons 1A 2U 

 and 2A 1U. 



The triplet code letters for tyrosine are 

 1A 2U. But is the actual sequence AUU, 

 UAU, or UUA? Short sequences of ribo- 

 tides (oligoribotides) can be lengthened at 

 their nucleoside (3') ends by polynucleo- 

 tide phosphorylase. A mixture of AUU 

 and AAU oligoribotides (the base at the 



8 As shown by T. Staehlin, F. O. Wettstein. H. 

 Oura, and H. Noll (1964). 



5' end is always written first in the se- 

 quence) is lengthened at the 3' end with 

 uridylic acid residues. When the length- 

 ened, mixed polyribotide — AUUU . . . U 

 or AAUUU . . . U — is tested for poly- 

 peptide synthesis, it is found that phenyl- 

 alanine and tyrosine are incorporated in 

 significant amounts and that no significant 

 amounts of isoleucine (which also has the 

 code letters 1A 2U) or of asparagine and 

 lysine (whose code letters are 2 A 1U) are 

 incorporated. Therefore, the code sequence 

 for tyrosine is probably AUU. 



Another method of attack for determining 

 base sequence in codons makes use of the 

 mutations causing single amino acid substi- 

 tutions in hemoglobin (see Figure 32-7. p. 

 415), TMV, tryptophan synthetase, and 

 other proteins. Those mutations occurring 

 spontaneously or with mutagens expected 

 to produce single base substitutions are as- 

 sumed to involve single base changes. In 

 TMV, a mutant causes tyrosine (AUU) to 

 be replaced by phenylalanine (UUU); the 

 mutant apparently causes a single base 

 change from A to U. In tryptophan syn- 

 thetase, a mutant replaces tyrosine (AUU) 

 by cysteine (GUU) and presumably involves 

 a change from A to G. In hemoglobin- 

 Mi.,,.,,,,,, the a chain (see p. 414) has the 

 histidine (1A 1U 1C) at position 58 changed 

 to tyrosine (AUU). If only a single base 

 change — from C to U — has occurred, then 

 the codon for histidine must start with A 

 and is either ACU or AUC. In hemoglobin 

 Zurich, the amino acid at position 63 is 

 changed from histidine (ACU or AUC) to 

 arginine (1G 1C 1U). This change is prob- 

 ably from A to G, so that the first base in 

 the arginine codon is G, and the codon is 

 either GCU or GUC. A continuation of 

 this kind of analysis has made it possible '•' 

 to assign complete U-containing nucleotide 

 sequences to the codons for nineteen of the 

 twenty amino acids. These sequences (listed 



9 For T. H. Jukes. 



