Effects of different extraction procedures on the molecular characteristics of bacterial ribosomal ribonucleic acid

Abstract

Ribosomal RNA has been extracted from Escherichia coli ribonucleoprotein, (RNP) in such a way as to obtain a maximal yield of 23-S particles. If RNP suspensions, with concentrations of more than 3 mg/ml, were rapidly treated with buffer-equilibrated phenol and bentonite, the relative amounts of 16-S and 23-S RNA obtained always corresponded to the relative amounts of 30-S and 50-S particles of the RNP originally present. Phenol extraction of RNP, in ionic environments varying from 0.05 M EDTA to 0.01 M MgCl 2 (in 0.01 M Tris buffer), gave 16-S and 23-S RNA in a constant ultraviolet absorbancy ratio of 1:2. As 23-S RNA is about twice the size of 16-S RNA, the numbers of each type of particle were therefore equal. Different results were observed if the concentration of the particles extracted with phenol was lowered to less than 0.1 mg/ml. When the Mg 2+ concentration, during phenol extraction of RNP, was less than 1 mM, the ratio of 16-S to 23-S RNA produced from equal numbers of 30-S and 50-S RNP particles changed irreversibly from 1:2 to 2:1. Extraction of RNP in the presence of 0.05 M EDTA gave almost entirely 16-S RNA. No ribosomal RNA species smaller than 16-S were obtained from E. coli RNP in any of the extraction procedures. Prolonged dialysis of any of the RNA specimens against 0.05 M EDTA and bentonite did not alter the ratio of 16-S to 23-S RNA originally observed, nor was there any further dissociation of the RNA into smaller subunits. Dialysis against 8 M urea in 0.05 M EDTA also had no effect on the original ratios. Similar results were obtained after the treatment of ribosomal RNA with 0.1 M periodate at pH 6.0 or with 0.1 M hydroxylamine at pH 7.0. The passage of ribosomal RNA through sodium amberlite columns was without effect if bentonite was also present. It is concluded that both 16-S and 23-S ribosomal RNA in E. coli are molecules without readily dissociable subunit structures. The isolation of 23-S RNA is more dependent than the isolation of 16-S RNA on changes in the ionic environment during phenol extraction. This is taken to indicate the presence in 23-S RNA of labile bonds which are unduly sensitive to such changes. The observation of two different effects on the amounts of 23-S and 16-S RNA obtained from bacterial RNP, depending on the concentration of RNP during phenol extraction, is taken as evidence against ribonuclease being responsible for the dissociation of 23-S RNA to 16-S in these experiments.