38. BIOSYNTHESIS OF PROTEINS IN BACTERIAL CELLS 445 



who have observed that it is possible to destroy RNA in vivo by adding 

 ribonuclease to intact cells of various types (onion roots, amoebae, etc.). 

 The effects of ribonuclease on bacterial RNA have been intensively studied 

 by Groth, 186 and by Jeener 187 with B. megaterium, by Jerne and Mal0e, 188 

 and by Francoise Gros 189 in E. coli. 



In amoebae, ribonuclease seems to penetrate according to a mechanism 

 of pinocytosis 190 and the amount of enzyme taken up depends on the ex- 

 external concentration, and can reach 2 X 10 11 molecules per cell. 



In certain species of bacteria such as B. megaterium, growth in a syn- 

 thetic medium is prevented by ribonuclease; others such as E. coli are sensi- 

 tive only when treated in the absence of salts. 188, 189 It has been found that 

 the action of ribonuclease on E. coli takes place in two stages: (1) formation 

 of a complex between ribonuclease and bacteria (and probably between 

 ribonuclease and RNA) ; and (2) destruction of the RNA. 



The first stage is inhibited by salts, whereas the second cannot proceed 

 in their absence. By adding ribonuclease to bacteria in distilled water and 

 by interrupting the first phase by salt addition, followed by incubation for 

 a sufficient period of time to permit the second phase to proceed, one can 

 easily follow the effect of ribonuclease on E. coli. Under these conditions, 

 the amount of RNA declines exponentially with the time of contact be- 

 tween enzyme and cells during the first phase. 



The metabolic activities of cells whose RNA has been partially or fully 

 destroyed have been extensively studied. In amoebae or in plants, partial 

 RNA destruction by ribonuclease does not affect the energy-yielding mecha- 

 nisms, 191 since neither the ATP content nor O2 consumption are sensibly 

 modified. Similar conclusions apply to bacteria 189 in relationship to their 

 capacity to oxidize succinate or glucose. In regard to the ability of syn- 

 thesizing proteins, it is observed that onion roots or amoebae, which 

 have lost their basophilia after ribonuclease treatment, are unable to in- 

 corporate C 14 -amino acids into protein. 191 



In E. coli, ribonuclease influences the ability to synthesize protein by two 

 distinct mechanisms. 189 Even after a very short time of contact with negli- 

 gible destruction of the RNA, an immediate drop of about 50 per 100 in 

 the ability to synthesize protein (as measured by S 35 incorporation) is ob- 

 served. When the incubation proceeds for a longer time a gradual destruc- 



186 D. P. Groth, Biochem. et Biophys. Acta 21, 18 (1956). 



187 R. Jeener, Biochim. et Biophys. Acta 32, 99 (1959). 



188 N. K. Jerne and O. Maaloe, personal communication (1958). 



189 Francoise Gros, unpublished data (1960). 



190 J. Brachet, M. Briers, L. Ledoux, A. Pileri, V. Schumaker, Y. Thomas, and F. 

 Vanderhaeghe, in "Radioisotopes in Scientific Research," Vol. 3, p. 285. Pergamon 

 Press, London, 1957. 



191 J. Brachet, "Biochemical Cytology." Academic Press, New York, 1957. 



