I i)() 2 |. 1). WAT SO N 



\ / \ / 



(0) N— AA, AA 2 AA 3 cf — "N AA 4 C 



i-r Oh o 



SRNA a SRNA b 



H ,° 



\ / 



(b) \ AA, AA 2 AA 3 AA 4 C5 + SRNA a 



SRNA b 



\ ig. s. Stepwise growth of a polypeptide chain. Initiation begins at the free NH 2 end 

 with the growing point terminated by a sRNA molecule. 



Since the immediate precursors arc amino-acyl-sRNA molecules, their re- 

 sult predicts that the polypeptide chain is terminated at its carboxyl growing 

 end by an sRNA molecule (Fig. 5). To test this scheme, we began some 

 studies to see whether sRNA bound specifically to ribosomes. Cannon and 

 Rjrug 49 tirst examined binding in the absence of protein synthesis. They 

 showed that in 10 2 MMg + f each 50s sub-unit of the 70s ribosome revcrsibly 

 bound one sRNA molecule. The same amount of reversible binding occurs 

 with amino-acyl-sRNA or with free sRNA and in the presence or absence 

 of protein synthesis. 



Protein synthesis, however, effects the binding observed in io -4 M Mg 4_+ . 

 In the absence of protein synthesis no sRNA remains ribosomal bound when 

 the Mg f+ level is lowered from 10 2 M to io~ 4 M. On the contrary, fol- 

 lowing amino acid incorporation, sRNA molecules become tightly fixed to 

 the « stuck » 70s ribosomes, whose nascent polypeptide chains prevent easy 

 dissociation to 30s and 50s ribosomes. One sRNA molecule appears to be 

 attached to each stuck ribosome. Prolonged dialysis against io~ 4 M Mg ++ 

 eventually breaks apart the stuck ribosomes. Then all the bound sRNA as 

 well as almost all the nascent protein is seen attached to the 50s component 

 supporting the hypothesis that these bound sRNA molecules arc directly 

 attached to nascent chains (Fig. 6). Direct proof comes from recent exper- 

 iments in which Gilbert 50 used the detergent duponol to further dissociate 

 the 50s ribosomes to their protein and RNA components. Then the nascent 

 protein and bound sRNA remained together during both sucrose gradient 

 ccntrifugation and separation on G200 Sephadcx columns. Following ex- 



s-126 



