Ill 



( IIM'TER 34 



long oligoribotides, for example, a chain of 

 500-1000 uridylic acids. Although poly U 

 greatly stimulates phenylalanine incorpora- 

 tion into protein, the single trinucleotide 

 UUU does not. Recall, however, that early 

 steps in protein synthesis require the activa- 

 tion and attachment of the amino acid to a 

 specific sRNA molecule. This "charged" 

 sRNA binds to the ribosome and, as directed 

 by the messenger RNA, is incorporated at 

 the end of the growing peptide chain. Poly 

 U causes Phe-sRNA to be bound to ribo- 

 somes; other polynucleotides cause other 

 specific charged sRNAs to be bound. 



One can synthesize or isolate oligoribo- 

 tides and test them for their in vitro ability 

 to bind specifically charged sRNAs to ribo- 

 somes. 1 " (According to convention, a tri- 

 ribotide of U with a 3'-terminal phosphate 

 is designated UpUpUp and one with a 5'- 

 terminal phosphate, pUpUpU.) When 

 pUpUpU, pApApA, and pCpCpC are 

 tested, they are found to direct the binding 

 of Phe-, Lys-. and Pro-sRNA, respectively; 

 dinucleotides have no effect. Moreover, 

 trinucleotides with 5'-terminal phosphate 

 are more active than those with no terminal 

 phosphate, and trinucleotides with 2'-(3')- 

 terminal phosphate are inactive. 



From other work 2U 1G is known to be a 

 code word for valine. The order of the 

 bases can be investigated using poly UG, 

 dinucleotides, the trinucleotide GpUpU, and 

 its sequence isomers UpGpU and UpUpG. 

 The binding of C 14 -Val-sRNA to ribosomes 

 is found to be directed both by poly UG and 

 GpUpU but not by UpGpU, UpUpG, or 

 dinucleotides. GpUpU has no effect upon 

 the binding of sRNAs, corresponding to 17 

 other amino acids, to ribosomes. There- 

 fore, we conclude from these results that a 

 code word for valine is GpUpU, and we 

 predict that a GUUGUUGUU . . . GUU 

 polymer will stimulate only valine incor- 



10 See M. Nirenberg and P. Leder (1964). and 

 P. Leder and M- W. Nirenberg (1964). 



poration into protein. Similar work showed 

 that UpUpG is a code word for leucine and 

 possibly UpGpU a code word for cysteine. 



Although there will undoubtedly be cor- 

 rections and additions to the codons in Fig- 

 ure 34-1 (some contradictory base sequence 

 results are obtained using the different in 

 vitro methods described ), examination of the 

 codons listed reveals a common feature to 

 some of the degeneracy already detected. 

 For example, two of the codons for leucine 

 have U at both ends. In other words, they 

 share the same doublet, so that their codons 

 can be written U • U, in which • can be A 

 or G. Although the base sequences in 

 alanine's two codons without U are postu- 

 lated, both contain a C and a G, as does 

 the U-containing sequence, so that one can 

 refer to a C • G shared doublet, in which • 

 probably can be U, A, or C. These and 

 other doublets are listed in the figure. The 

 meaning of such shared doublets in the de- 

 generate in vitro RNA code is not yet clear, 

 nor is it known to what extent triplets with- 

 out U code in messenger RNA in vivo. 



That the frequency of some of the amino 



acids in protein remains nearly constant 



A 4- T 

 when there are large shifts in the — — !— — 



C -f- G 



ratio is evidence for the existence of degen- 

 eracy in vivo. Leu-sRNA of E. coli can be 

 separated into three types, each with dif- 

 ferent coding properties in vitro. 11 The first 

 type responds preferentially to poly UC, the 

 second type responds to poly U and copoly- 

 mers rich in U (including poly UC), and 

 the third responds preferentially to poly UG. 

 The discovery that leucine is carried by dif- 

 ferent sRNAs provides an explanation for 

 the observations in vitro that the coding unit 

 for leucine is degenerate ( at least four dif- 

 ferent triplets serve to encode it ) and that 

 the UUU codon is ambiguous (since it is a 

 codon for both leucine and phenylalanine). 

 Assuming that there is only one DNA locus 



11 See G. von Ehrenstein and D. Dais (1963). 



