VII. SYNTHESIS OF RNA AND RIBOSOMES 333 



sequently released as soluble protein (see Section I,E). In addition, that 

 part of the newly formed protein which is destined to be a part of the 

 ribosomes does not appear to be firmly bonded to the RNA because a 

 considerable proportion is not eluted from DEAE. Thus chromatography 

 cannot be used to complement the results of sedimentation analysis and 

 the sedimentation analysis is obscured by the presence of nascent protein. 



The time course of incorporation of C^^-leucine is shown in Fig. 24, 

 taken from Britten et al. (1962). Figure 25 shows the sedimentation 

 profiles obtained at various times after a 1 -minute period of incorpora- 

 tion was terminated by dilution of the tracer. 



The results differ markedly from those obtained with uracil as a 

 tracer. There is no sign of the delay which would be apparent if a con- 

 siderable pool of precursors to ribosome protein existed. Most of the 

 C"-leucine radioactivity appears to enter the SOS and 50S ribosomes 

 directly. In addition, a part of the tracer enters the 43S neosome and is 

 subsequently transferred to the SOS ribosome. 



B. EFFECT OF CHLORAMPHENICOL ON RIBOSOME SYNTHESIS 



Chloramphenicol, which inhibits protein synthesis, provides a sub- 

 sidiary technique for showing which stages of ribosome synthesis depend 

 on the addition of protein. Figure 26 shows that in the presence of 

 chloramphenicol most of the C"-uracil tracer is restricted to particles 

 having sedimentation coefficients less than 20S. This RNA has been 

 shown by Bolton (1959) to have ribosomal base composition. 



C. ASSEMBLY OF THE RIBOSOMES 



The experiments reported relating to the synthesis of ribosomal 

 protein do not add any fundamentally new features to the sequence of 

 ribosome synthesis. They do, however, confirm other indications that 

 some of the steps in the sequence represent the addition of protein. The 

 fact that a high proportion of the leucine label enters 30S and 50S 

 ribosomes directly with delays of less than 1 minute shows that the last 

 stages of synthesis (30S neosome^ 30S ribosome and 43 neosome-^ 508 

 ribosome) involve the addition of protein. This is in agreement with the 

 fact that no new RNA is incorporated directly into ribosomes and with 

 the low protein RNA ratio of neosomes as indicated by their column 

 behavior. 



One other feature is clear from the leucine pulse and chase experi- 

 ments. Since the 43S receives some label at early times it is evident that 

 not all the protein of the 50S is added in one step. In fact, the chase 

 experiment shows that the 50S increases some 30% in specific radioac- 

 tivity during an 8-minute chase period at the expense of the 43S. Rough 



