Synthesis and maturation of ribosomal RNA in Escherichia coli

Abstract

A high resolution autoradiographic technique has been developed for the quantitative analysis of the distribution of 14C-labeled RNA on polyacrylamide gels after electrophoresis. We have used this technique to study the synthesis and maturation of ribosomal RNA in Escherichia coli and have obtained the following results: 1. (1) There exist one or perhaps two precursors to the 16 s RNA and one detectable precursor to the 23 s RNA, each of slightly lower electrophoretic mobilities than the mature forms. The precursor-product relationships are proven by accurate kinetic experiments, pulse-chase experiments and, most convincingly, by the demonstration that maturation takes place when further RNA synthesis is inhibited by actinomycin D. 2. (2) Protein synthesis inhibition by chloramphenicol treatment or methionine starvation of stringent cultures inhibits ribosomal RNA maturation and causes the accumulation of large amounts of these precursors. The precursor RNA which accumulates in methionine-starved cultures is converted to the mature forms when methionine is restored. 3. (3) The kinetics of synthesis of 5 s ribosomal RNA are essentially first order and hence do not provide evidence for the existence of a pool of a macromolecular precursor for the 5 s. This is corroborated by experiments which show that there is only a negligible increase in label in 5 s RNA if actinomycin D is added to a culture pulsed for two minutes with RNA precursors. 4. (4) A 5 s-like RNA of slightly lower electrophoretic mobility than normal mature 5 s RNA accumulates in chloramphenicol-inhibited or methionine-starved cultures. This species also matures upon restoration of methionine to the starved culture. This species is not produced by nucleolytic cleavage of one of the high molecular weight ribosomal RNA precursors during its maturation since the synthesis of this 5 s-like RNA, is not coupled with the maturation of 23 or 16 s RNA in either normal or inhibited cultures. No other precursor to the 5 s RNA can be detected in E. coli during exponential growth or during protein synthesis inhibition. 5. (5) Ribosomal RNA is synthesized during the period of recovery from methionine starvation, contrary to the conclusions of Nakada, Anderson & Magasanik (1964). However, there is a long delay in the appearance of label in mature 16 s ribosomal RNA, presumably due to dilution of the newly-synthesized radioactive precursor RNA by the “relaxed” RNA accumulated during the starvation period.