BIOSYNTHETIC PRECURSORS OF 30S AND 50S RIBOSOMAL PARTICLES IN ESCHERICHIA COLI

The biosynthesis of Escherichia coli 30S and 50S ribosomes was studied, making use of exponentially growing fragile cultures. These cultures permit rapid lysis and total recovery of cellular ribonucleic acid (RNA). By analysis of lysates in zonal sedimentation in sucrose gradients, it was found that mature ribosomes appear in polyribosomes and free 30S and 50S ribosomal particles, while the newly formed ribosomal ribonucleic acid (rRNA) en route to complete ribosomes does not appear in polyribosomes to a measurable extent for at least 6 min (doubling time, 120 min; Mangiarotti, G., and Schlessinger, D. (1966a), Nature 211, 761; (1966b), J. Mol. Biol. 20, 123; (1967), J. Mol. Biol. 29, 365). To analyze the synthesis of ribosomes, [3H]uracil was added to the cultures, and samples were lysed and fractionated in sucrose gradients to follow the flow of 3H into RNA. The newly synthesized rRNA appears in precursors that sediment more slowly than complete ribosomes. A whole array of material is observed sedimenting less than 26 S; the labeled RNA extracted from this region is identified as incomplete chains of 16S and 23S rRNA, since it sediments slower than 16S, but competes with purified unlabeled 16S and 23S rRNA in deoxyribonucleic acid-ribonucleic acid hybridization. Since RNA from this region sediments more slowly after phenol extraction, proteins are probably attached to the growing rRNA chains. This conjecture is supported by the observation that about one-half the protein moving in this region of the gradients no longer sediments there after brief treatment of lysates with RNase. The proteins released resemble ribosomal proteins in their behavior on elution from carboxymethylcellulose columns. Three well-defined peaks of larger precursors, sedimenting at 26, 32, and 43 S, were also characterized. RNA extracted from the 26S peak contained only 16S RNA, so that it was assigned as a precursor of the 30S ribosome. The 32S and 43S particle were judged to be successive stages in the maturation of the 50S ribosome, since they contained 23S RNA. The kinetics of labeling of complete rRNA chains indicate that they are formed in 2 min (13 nucleotides/sec) in these cultures. To produce the observed numbers of chains, a number of RNA polymerase molecules must be working in tandem on each cistron specific for rRNA. About 0.5% of the total rRNA is in unfinished chains at any instant. Once completed, 16S rRNA chains are arrested as 26S precursors for an average of about 18 min before they mature to 30S ribosomes; the pool of 26S material contains about 3.5% of the total rRNA. The 23S rRNA halts about 1 min as a 32S precursor, in a pool containing about 0.4% of the total cellular rRNA. The 32S particle then becomes a 43S precursor, in a pool containing about 7% of the cellular rRNA, for about 17 min more before maturing to a 50S ribosome. Thus, complete 30S and 50S ribosomes are made in the same over-all time. © 1968, American Chemical Society. All rights reserved.