Studies on the genesis of polyribosomes: I. Origin and significance of the subribosomal particles

Abstract

In addition to polyribosomes and 74 s ribosomal monomers, there exist in the cytoplasm of HeLa cells two classes of subribosomal particles, namely, 60 s particles which contain 28 s RNA, and 40 s particles which contain 16 s RNA. Evidence is presented that their presence in cytoplasmic cell fractions is not a consequence of dissociation of 74 s ribosomes due to a deficiency of magnesium ions after cells are disrupted. 60 s and 40 s particles are always present in strictly equivalent numbers and together account for about 10% of total ribosomal material in the cytoplasm. In contrast, the number of free 74 s ribosomal monomers may vary widely (although it is strictly constant when aliquots of the same cell suspension are examined); on occasions, cytoplasmic fractions containing more 60 s particles than 74 s ribosomes have been observed. However, the total number of 74 s ribosomes (“polymers” plus monomers) within the cell tends to be fairly constant. The smaller the number of free 74 s ribosomes, the larger is the number released from polyribosomes by treatment with puromycin. Such treatment, however, does not change the number of 60 s and 40 s particles, nor do 74 s ribosomes released from polyribosomes dissociate detectably to 60 s and 40 s particles within 60 minutes. Labeling with [ 14 C]uridine has shown that 60 s and 40 s particles are made in the nucleus and enter the cytoplasm as individual entities, the latter preceding the former. Combination of 60 s particles with 40 s particles to form 74 s ribosomes proceeds mainly, and most probably exclusively, in polyribosomes, that is, while the 40 s particle is attached to a messenger RNA molecule. The relative specific radioactivity of 74 s ribosomes in polyribosome form often exceeds that of free 74 s ribosomal monomers for at least 3 hr after synthesis of the constituent subribosomal particles. The population of free 74 s ribosomal monomers exchanges only slowly with the population of 74 s ribosomes in polyribosome form, the rate depending on the metabolic state of the cell. Those 74 s ribosomes which are liberated from the messenger RNA molecule by puromycin recombine with it preferentially on removal of puromycin. This is interpreted as a topographical effect; other explanations are possible. The implications of these findings are discussed.