432 



CHAPTER 47 



ribosomes. (Guanosine triphosphate is re- 

 quired in order that the labeled amino acids, 

 carried by transfer RNA, appear attached 

 to the ribosome.) 



It can be hypothesized (as illustrated in 

 Figure 47-2) that each of the two DNA 

 strands (which are complements) specifies 

 an RNA complement (the two RNA strands 

 would then also be complementary and com- 

 prise informational or template RNA). 

 Large segments of one of the informational 

 RNA templates leave the nucleus as mes- 

 senger RNA and become located in already 

 formed ribosomes, while the complementary 

 RNA template is broken into segments which 

 are used to make transfer RNA and possibly 

 other RNA's (such as ribosomal RNA). In 

 this event, transfer RNA and messenger 

 RNA would be complementary, they could 

 pair at the ribosome in the cytoplasm, and, 

 in doing so, would place the transported 

 amino acids in proper sequence. These 

 amino acids could then be enzymatically 

 joined to form a polypeptide, which could be 

 freed from transfer RNA, which, in turn, 

 could be liberated from pairing with its 

 messenger complement. 



The mechanism of protein synthesis can 

 be studied using a suspension of ruptured 

 cells. Such a cell-free system can be pre- 

 pared from E. coli. The activity of the cell- 

 free system can be preserved by the addition 

 of mercaptoethanol. Also added to the mix- 

 ture are the triphosphates of the ribo- 

 nucleosides of A, G, C, and U, as well as 

 all 20 of the amino acids in their L forms. 

 The synthesis of protein can be readily de- 

 tected if one of the added amino acids is 

 radioactive. If the labeled amino acid is 

 valine, for example, valine is found to be- 

 come incorporated into protein. This in- 

 corporation can be stopped by the addition 

 of DNAase, which destroys the DNA, 

 thereby halting the production of messenger 

 RNA. In the absence of new messenger 

 RNA, protein synthesis stops. 



That this effect of DNAase concerns the 

 production of messenger RNA is demon- 

 strated (1) by the absence of valine incorpora- 

 tion when sRNA or ribosomal RNA is added 

 to the system, and (2) by the resumption of 

 valine incorporation when the RNA from 

 washed ribosomes is added to the system. 

 The informational RNA added may come 

 from various sources. Some experiments 

 have studied the in vitro conditions for the 

 synthesis of /3-galactosidase ^ (see Chapter 

 46), using messenger RNA obtained from 

 genetically different induced and noninduced 

 E. coli. It is even possible to add TMV RNA 

 to the bacterial cell-free system and detect 

 the synthesis of TMV protein.*^ 



Using a bacterial cell-free system it is 

 also possible to study whether the addition 

 of synthetic polyribonucleotides has any ef- 

 fect on protein synthesis. First, pure poly- 

 ribotides containing only one base. A, or C, 

 or U, are added. The first two fail to result 

 in amino acid incorporation. However, the 

 addition of polyuridylic acid causes 

 L-phenylalanine to be incorporated into 

 protein.^ It is found, moreover, that the 

 protein formed is poly-L-phenylalanine, and 

 that no other amino acid is incorporated. 

 It is also found that phenylalanine linked to 

 sRNA is an intermediate in this process. 

 This surely means that wherever an appro- 

 priate sequence of U's appears in normal 

 messenger RNA, the protein being synthe- 

 sized will incorporate L-phenylalanine. This 

 is the first crack in the RNA code, that is, 

 the first determination of a sequence of mes- 

 senger RNA nucleotides which specifies the 

 incorporation of a particular amino acid into 

 protein. Note that the problem of DNA 

 coding, mentioned on page 427, has become 

 a problem of RNA coding. 



The results mentioned also tell us that 

 certain ribonucleotide sequences do not have 



In work of G. D. Novelli and coworkers. 

 M, W. Nirenberg and coworkers. 



