(26 



( n \i' 1 1 i< 33 



Messenger RNA can be made using DNA 

 located in the cytoplasm as template; for 

 instance, the DNA of the vaccinia virus in 

 human-tissue culture cells.' 1 



mRNA Synthesis 



How is messenger RNA synthesized? DNA 

 can replicate in vitro in the absence of RNA; 

 such a replication probably also occurs in 

 the nucleus, although there might be subtle, 

 secondary interactions with RNA or protein. 

 The evidence mentioned proves that mRNA 

 synthesis is intimately related to DNA. It 

 is found ' that the nucleus normally contains 

 an enzyme, DNA-dependent RNA poly- 

 merase, necessary for RNA synthesis. This 

 enzymatic RNA synthesis can be performed 

 in vitro and requires the presence of DNA 

 as well as all four riboside triphosphates. 

 Under certain conditions, the RNA synthe- 

 sized has the same base ratio as its DNA 

 primer-template (except, of course, that T 

 is U). This situation is reminiscent of the 

 synthesis of DNA, in which the DNA poly- 

 merase is directed by single-stranded DNA. 

 In vitro, however, double-stranded DNA is 

 a more effective primer in making RNA 

 polymer than single-stranded DNA. 



The DNA in mature <£X174 is in the form 

 of a single-stranded ring. In E. coli, <£X174 

 in its replicative form (RF) is a double- 

 stranded, circular, DNA helix. If the double 

 ring is broken. RNA polymerase in vitro 

 makes RNA complementary to both single- 

 stranded DNA rods. If, however, the phage 

 DNA is extracted carefully so that the circle 

 or ring is not broken, the results indicate 

 that only one of the two DNA strands will 

 serve as template. Moreover, it is found 

 in vivo as well as /'// vitro that the DNA circle 

 serving as template for RNA polymerase is 

 the same one used to produce the single- 



,; See Y. Becker and W. K. Joklik (1964). 



7 From the work of J. Hurwitz. of A. Stevens, of 



S. B. Weiss, their colleagues and others. 



stranded DNA found in the mature phage." 

 This finding not only proves the occurrence 

 of one-complement transcription but seems 

 to indicate that the control mechanism in this 

 case at least requires the circular DNA 

 double helix to be intact. 



Not all RNA is made from a DNA tem- 

 plate. We have already noted (p. 366) 

 that, in vivo, viral RNA is used as a template 

 by RNA synthetase (RNA-dependent RNA 

 polymerase) to make complementary RNA. 

 Moreover, in vitro, homopolyribotides of A, 

 U, and C can also serve as templates in the 

 synthesis of complementary RNA by an 

 RNA polymerase/' 



At low concentrations, actinomycin D 

 binds with G-containing sites in DNA, thus 

 selectively suppressing the synthesis of mes- 

 senger RNA by RNA polymerase. Inter- 

 ference with DNA synthesis also occurs but 

 at much higher concentrations. Experi- 

 mental evidence suggests 10 that: 



1. Actinomycin lies in the minor groove 

 of DNA 



2. The minor groove is the specific tem- 

 plate site for DNA-dependent RNA 

 polymerase 



3. The major groove is the site for DNA 

 polymerase action. 



Ribosomal RNA 



Since the typical chromosome of higher or- 

 ganisms contains RNA, some of the newly- 

 synthesized RNA appears in the nucleus as 

 part of parent and daughter chromosomes. 

 Most of the newly-made RNA, however, 

 leaves the chromosomes, and a considerable 

 portion is presumably used in the manufac- 

 ture of new ribosomes. Studies with Bacillus 



* See M. Hayashi, M. N. Hayashi, and S. Spiegel- 

 man (1963. 1964). B. Chandler, M. Hayashi. 

 M. N. Hayashi. and S. Spiegelman ( 1964), and 

 M. H. Green ( 1964). 

 '■' See J. S. Krakow and S. Ochoa (1963). 

 10 See E. Reich (1964). 



