Kaplan R, Apirion D
J Biol Chem. 1975 Mar 10;250(5):1854-63.
The disappearance of ribosomes in Escherichia coli cells starved for a carbon source was studied. We used a series of mutants, some of them lacking in ribonuclease I(RNase I, EC 2.7.7.17), and other containing various combinations of modified polynucleotide phosphorylase (PNPase, EC 2.7.7.8) and modified ribonuclease II (RNase II, EC 3.1.4.1). RNA was prepared from the starved mutant cells and separated on polyacrylamide gels. The results obtained indicate that 23 S RNA degradation is similar in all strains that lack RNase I, and is slightly increased in the strain that contains this enzyme. The extent of 16 S RNA degradation is identical in all strains tested. RNA species in the size of 4 S and smaller accumulate in mutants containing modified forms of PNPase and RNase II. The appearance of an RNA species 10% smaller than 16 S RNA (d16 S RNA) was observed in all strains that contain unmodified RNase II. Analysis of ribosomes and polysomes and their RNA content indicated that polysomes are converted to monosomes and these, in turn, to ribosomal subunits. No RNA degradation products were found in polysomes, 70 S, OR 50 C particle; 30 S subunits contained 16 S RNA as well as the d16 S RNA species. Subunits are degraded to a similar extent in all strains lacking RNase I, and at a slightly faster rate in the strain that contains RNase I. The RNA to protein ratio in subunits prepared from starved cells is similar to that of unstarved cultures. Very little degradation of ribosomal proteins occurs in these mutants during carbon starvation. The proteins released from degraded ribosomes are found in the fast sedimenting (20,000 times g) pellet. Cell viability studies indicated a direct correlation between the capacity of the mutants to recovery from starvation and their capacity to degrade RNA. Thus a biological necessity for degradation of ribosomes during starvation is implied. Based on these data we propose that the endonucleolytic degradation of ribosomal RNA is the primary event in starvation degradation. It takes place in ribosomal subunits, which fall apart after the endonucleoltic attack. The RNA pieces produced by this cleavage are degraded to nucleotide by RNase II and PNPase. The ribosomal proteins attach to the cell membrane.
对缺乏碳源而饥饿的大肠杆菌细胞中核糖体的消失情况进行了研究。我们使用了一系列突变体，其中一些缺乏核糖核酸酶I（RNase I，EC 2.7.7.17），另一些含有修饰的多核苷酸磷酸化酶（PNPase，EC 2.7.7.8）和修饰的核糖核酸酶II（RNase II，EC 3.1.4.1）的各种组合。从饥饿的突变体细胞中制备RNA，并在聚丙烯酰胺凝胶上进行分离。所得结果表明，在所有缺乏RNase I的菌株中，23 S RNA的降解情况相似，而在含有该酶的菌株中降解略有增加。在所有测试菌株中，16 S RNA的降解程度相同。在含有修饰形式的PNPase和RNase II的突变体中，4 S及更小尺寸的RNA种类会积累。在所有含有未修饰RNase II的菌株中，均观察到一种比16 S RNA小10%的RNA种类（d16 S RNA）。对核糖体和多核糖体及其RNA含量的分析表明，多核糖体转化为单核糖体，进而转化为核糖体亚基。在多核糖体、70 S或50 C颗粒中未发现RNA降解产物；30 S亚基含有16 S RNA以及d16 S RNA种类。在所有缺乏RNase I的菌株中，亚基的降解程度相似，而在含有RNase I的菌株中降解速度稍快。从饥饿细胞中制备的亚基中RNA与蛋白质的比例与未饥饿培养物中的相似。在这些突变体中，碳饥饿期间核糖体蛋白的降解很少。从降解的核糖体中释放的蛋白质存在于快速沉降（20,000倍重力）的沉淀中。细胞活力研究表明，突变体从饥饿中恢复的能力与其降解RNA的能力之间存在直接相关性。因此，暗示了饥饿期间核糖体降解的生物学必要性。基于这些数据，我们提出核糖体RNA的内切核酸酶降解是饥饿降解中的主要事件。它发生在核糖体亚基中，在核酸内切酶攻击后亚基解体。这种切割产生的RNA片段被RNase II和PNPase降解为核苷酸。核糖体蛋白附着在细胞膜上。
